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N A L A R T I C L E

Hypoglycemic Activity ofGlyburide Glibenclamide)Metabolites in HumansTONY RYDBERG, MS C PHARMANDERS JONSSON, MD

MICHAEL RODER, MDARNE MELANDER, MD, PHD

O B JE C T IV E To assess the hypoglycemic effect and th e insulin-releasing effect ofthe main glyburide (glibenclamide) metabolites 4-trans-hydroxy-glibenclamide (Ml)and 3-cis-hydroxy-glibenclamide (M2) in humans.RESEARCH DESIGN AND METHODS Eigh t he a l thy sub je c t s pa r t i c i -pated in a placebo-con trolled, ran dom ized, single-blind crossover study with fivesingle-dose tests, 3 months apart: 3.5 mg glibenclamide (Gb) orally, 3.5 mg Gb intra-venously, 3.5 mg Ml intravenously, 3.5 mg M2 intravenously, and placebo intrave-nously, each in the fastingstate.Standardized meals were given 0.5 and 5.5 h after eachmedication. Blood glucose levels were measured by a glucose oxidase method, andserum insulin concentrations were analyzed by a specific immunoassay.R ES U L TS Blood glucose levels during the first 5 h were significantly lowered no tonly by Gb but also by Ml and M2. The mean SE blood glucose reductions (versusplacebo) expressed as percent of area un der th e curve (AUC) (0- 5 h) were 18.2 3.3%for M l, 12.5 2.3% forM 2,19.9 2.1%for intravenousGb,and 23.8 1.2% for Gborally. Serum insulinlevelswere significantly increased byGb aswell as by Ml andM2.C O N C L U S IO N S The two main metabolites of glyburide (glibenclamide) have ahypoglycemic effect in humans, which is due to increased insulin secretion.

lyburide (glibenclamide), the mostcommonly used sulfonylurea, iscompletely metabolized b y the liverand primarily eliminated as hydroxylatedderivatives, which are excreted in urine

and feces in similar amounts (1-4). Themain metabolites are 4-trans-hydroxy-glibenclamide (Ml) and 3-cis-hydroxy-glibenclamide (M2) (1,2,5). In humans,Ml and M2 are formed approximately in

From the Hospital Pharmacy(T.R.),Kristianstad Cou nty C entral Hospital, Kristianstad, Sweden;the Departments of Endocrinology (A.J.) and Clinical Pharmacology and Community HealthSciences (T.R., A.M.), Lund University, Malmo General Hospital, Malmo, Sweden; and the StenoMemorial Diabetes Center (M.R.), Gentofte, Denmark.

Address correspondence and reprint requests to Tony Rydberg, MSc Pharm, the HospitalPharmacy, Central Hospital, S-291 85 K ristianstad, Sweden.

Received for publication 7June 1993 and accepted in revised form 24 March 1994 .M l, 4-trans-hydroxy-glibenclamide; M2, 3-cis-hydroxy-glibenclamide; Gb, glibenclamide;

BM1,body mass index; ANOVA, analysis of variance; AUC, area unde r the curve.

the proportions (M1:M2) 3:1 to 6:1(1,2,4,6-8).

The hypoglycemic activities of M land M2 are allegedly low or nil(1,2,4,9,10), and this message has beenwidely spread in articles and transferredbetween several reviews (4,11 -22 ). How-ever, this view is derived from stud ies us-ing oral administration, from animalstudies, or by inference (1,2,5,9,23-25).Both M l and M2 are more polar than gli-benclamide (Gb) itself (3,6) and hencemay not be well absorbed from the gas-trointestinal tract. Therefore, parenteraladministration in humans would be nec-essary to assess their hy poglycemic activ-ity. This study examined the hypoglyce-mic and insulin-releasing activity of Mland M2, after intravenous administrationin healthy volunteers, and compared itwith that of the parent compound andwith placebo. Both metabolites werefound to possess considerable hypoglyce-mic and insulin-releasing capacity.

R E S E AR C H D E S I G N A N DM E T H O D S E ig ht n o n sm o k in g ,Caucasian volunteers (4 men, 4 women)were studied. They had a mean SD ageof 25.4 4 . 1 years at inclusion. Theirmean weight and body mass index (BMI)were 69.7 8.4 kg and 22.8 1.7 kg/m 2, respectively. After 1 year (e.g., at endof study) BMI was 22.7 2.1 kg/m 2. Allsubjects were healthy according to clini-cal status and routine laboratory analysesof hematologic, hepatic, and renal func-tion. Tests were postponed if hemoglobinlevels were

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dom order with 3-month intervals. Thetests included 3.5 mg intravenous Ml,3.5 mg intravenous M2, 3.5 mg intrave-nousGb,3.5 mg oral Gb, and intraveno usplacebo. Gb, Ml, and M2 were suppliedin powder form by Hoechst GmbH(Frankfurt, Germany ). Because Gb and itsmetabolites are almost insoluble in solu-tions with low or neutral pH, a sterilephosphate buffer 0.1 M, pH 10.0, 300mosm/1,was used, prepared at the Hospi-tal Pharmacy, Lund University Hospital(Lund, Sweden), according to existingGMP-rules. Because of reported hydroly-sis of sulfonylurea drugs at high pH (26),0.5 mg/ml solutions for injection wereprepared by a pharmacist immediatelybefore injection. T he dry pow der was dis-solved during 5-10 min in phosphatebuffer, and the solution was then filteredthrough a 0.2-jum sterile and pyrogen-free disposable filter (Filtron, Germany).From each preparation, a sample wassaved and frozen for later analysis of theactual concentrations of the solutions andto check possible loss in the filter. Con-centrations ofM l,M 2, and Gb were mea-sured by liquid chromatography (6).Seven milliliters of the phosphate bufferserved as placebo, and for oral Gb admin -istration, one 3.5mgDaonil tablet (HoechstGmbH, lot no . 257B246) was given.

All studies were started at 0745after overnight fasting (^10 h). Bloodsamples for hemoglobin, glucose, and in-sulin determinations were drawn. A cath-eter was inserted into an antecubital veinfor continuous infusion of 500 ml salinefor1 h. At 0800 ,abolus injection of7mldru g solution was given over 2 min via anindwelling cannula inserted intoavein onthe contralateral arm (except when Gbwas given orally). Simultaneously, thesubject drank 200 ml of tap water. Imme-diately after the injection, the catheterand vein w ere flushed with 5 m l saline toavoid occlusion of catheter and thrombo-phlebitis from the high pH of the injectedsolution.

Oral glucose was available to begiven if blood glucose concentration s fellbelow 1.0 mM or if severe hypoglycemic

symptoms occurred. After 30 min, thesubject had a standardized breakfast withan energy content of 1,800 kj (430 kcal)and after 5.5 h, a standardiz ed lu nch w ithan energy content of 3,150 kj (750 kcal).Apart from the standard meals and theinitial 200 ml of water, no food or liquidintake was allowed. Venous blood sam-ples (5 ml) for analyses of blood glucoseand serum insulin were drawn at regularintervals between 0 and 10 h. Blood glu-cose concentrations were determined byaglucose oxidase method on a Greinermultichannel analyzer. Serum insulinconcentrations were measured by a spe-cific enzyme-linked immunosorbent as-say (27). The detection limit was 5 pM,with an interassay coefficient of variationof 8.6%. Proinsulin, split (32-33)- anddes(31-32)-proinsulin did not cross-react in this assay.Statistical analysisData are shown as means SE,except forconcentrations of solutions for injections(mg/ml) and mean given doses (mg),which are presented as means SD. In-sulin data are also presented as mediansbecause of non-Gaussian distribution ofvalues. Area comparisons between pla-cebo and active treatment were analyzedby paired, two-tailed Student's ttest andrepeated measures analysis of variance(ANOVA). Other statistical comparisonsbetween group means were made withone-way ANOVA and post-testing by theBonferroni m ethod. N onparametric, two-tailed Wilcoxon's rank-sum tests wereused for comparisons of medians (pairedfor area comparisons and one sample testfor percen t effect com parisons) . The areasunder the blood glucose and serum insu-lin concentration curves (AUC, 0-5 h)were calculated after created cubic splines(smoothed) curves using GraphPad In-Plot software, version 4.03. The medianswere calculated using GraphPad InStatsoftware, version1.10a.R E S U L T S As shown in Fig. 1, notonly intravenous and oral glibenclamidebut also intravenous Ml and M2 reduced

blood glucose levels during the first 5 hafter administration. With one exceptionfor M2 (subject 5), both metabolites re-duced blood glucose in each of the 8 in-dividuals du ring the first 5 h after ad min-istration. All reductions were statisticallysignificant (P < 0.01) (Table 1). Insulinlevels were significantly increased (versusplacebo) by Gb as well as by Ml and M2(P < 0.05, Wilcoxon's rank-sum test)(Table 2).

Little or no effect on b lood glucoseand serum insulin levels could be dis-cerned after lunch (5-10 h). The bloodglucose reduction after intravenous Gbwas faster than that of oral Gb, but it alsowaned more quickly (Fig. 1). However,this finding could also relate to differencesin the effective doses; the liquid chro-matographic analyses of the injection so-lutions revealed losses of Ml, M2, and,particularly, Gb, probably because of ad-sorption or precipitation in the sterile fil-ter used immediately before injection.The mean SD concentra tions (n - 8)were 0.483 0.018 mg/ml (Ml), 0.470 0.047 mg/ml (M2), and 0.344 0.033mg/ml (Gb). The mean resultant doses SD were 3.38 0.13 mg for M l, 3.29 0.28 mg for M2, and 2.41 0.26 mg forintravenous Gb. The resultant doses perkilogram body weight are given in Table3,and it emphasizes that the intravenous(but no t the oral) Gb dose was lower thanthose of the two metabolites.

The relative reduction of bloodglucose and the relative increase of insu-lin subsequent to Gb and the metaboliteswere calculated asperc ent effect = 100

(AUC placebo - AUC treatment)X AUC placeboThe mean SE percent effects (reduc-tions of blood glucose) are given in Table1. The effects between metabolites andthe parent compound did not differ sig-nificantly except for M2 versus oral Gb(Bonferroni P < 0 .05). For serum in sulin,median values are also given in Table 2.

DIABETES CARE, VOLUME 17, NUMBER 9, SEPTEMBER 199 4 1 0 2 7

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Hypoglycemic glyburide metabolites

Figure Ejects of intravenously administered M l, M2, and Gb on blood glucose levels during the first 5 h after administration mean SE , n = 8). Astandard breakfast was ingested after 0.5 h. For comparison, Gb was also administered orally. O ,placebo;D , intravenous M2; A , intravenous Ml; M, oralGb ; , intravenous Gb.

The median percent effects were allsignif-icantly greater than zero (P < 0.05).C ON C LU SI O N S The resul ts showthat bot h m ain metabolites of Gb are ableto promote increased insulin secretionand pronounced hypoglycemia in hu-mans.Based on the mean percent red uc-

tion in blood glucoseAUCduring the first5 h relative to the mean resultant doses,M2 would have 30% less activity thanM l.Accurate comparisons with the par-ent compound are difficult, as the result-ant dose ofGbintravenously was reducedbecause of loss of the compound in thesterile filter used immediately before in-

jection. However, the resultant doses ofMl and M2 did not essentially differ fromthat of the parent dru g given orally (if thetablet content did not differ from thenominal 3.5 mg), suggesting that Mlwould have -75% and M2 -50% of thehypoglycemic activity of the parent com-pound (the 3.5 mg tablet is assumed to

Table 1Individual 0-5 h areas under the blood glucose concentration curves and corresponding blood glucose reductions as percent ofAUC 0-5 h) versus placebo

Subject no.12345678Mean SE

Placebo19.0523.7521.5820.4621.3319.7519.3421.15

20.80 0.54

AUC 0-5 h (mmolhIntravenous

M l12.8117.5319.6517.3117.9018.8514.3917.80

17.03 0.81

IntravenousM2

16.6918.9719.5217.2221.5418.1515.9217.77

18.22 0.63

r 1Intravenous

Gb16.3717.3018.9915.3917.5915.2116.3815.76

16.62 0.45

Oral Gb14.2416.7817.8615.2816.6715.0214.7416.16

15.84 0.43

IntravenousM l

32.826.2

8.915.416.1

4.625.615.8

18.2 3.3

Percent effectIntravenous

M212.420.1

9.515.80.08.1

17.716.012.5 2.3

IntravenousGb

14.127.212.024.817.523.015.325.5

19.9 2.1

Oral Gb25.229.317.225.321.823.923.823.6

23.8 1.2

1028 DIABETES CAR E, VOLUME 17, NUMBKR 9 , SEPTEMBER 199 4

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Table 2Individual 0-5 h areas under the serum insulin concentration curves and corresponding serum insulin increases as percent ofAUC 0-5 h) versus placebo

A U C O - S M p m o l - h - r 1 ) Percent effect

Subject no . Placebo IntravenousM lIntravenous

M2Intravenous

Gb Oral GbIntravenous Intravenous Intravenous

Ml M2 Gb Oral Gb12345678Mean SE 303.8 43.7 422.6 53.7 420.8 59.7 531.4 105.4 808.1 130.8 46.1 16.0 44.7 17.5 94.0 52.8 177.0 48.9Median 274.7 385.7 377.4 461.5 787.9 31.5 22.9 46.3 111.3

233.7328.5216.7315.7220.8581.8325.2208.6

311.2409.2518.7378.3393.1754.4296.6319.4

276.8518.8532.1318.5409.3740.5345.4224.7

368.5494.71,215

428.3299.4615.6533.4296.3

497.1572.81,2711,003

443.71,2211,073

383.1

33.224.6139.4

19.878.029.7

- 8 . 853.1

18.457.9145.5

8.985.427.3

6.27.7

57.750.6460.7

35.735.6

5 .864.042.0

112.774.4486.5217.7101.0109.9230.0

83.7

have complete bioavailability in contrastto the 5 m g tablet marketed in the U.S.).These results strongly contrastprevious reports that have claimed thatthe Gb m etabolites have little or no hypo-glycemic activity (1,2,5,9,23-25). How-ever, those conclusions were based onstudies using oral administration (1,2) oranimal studies (5,9,23,24) or on infer-ence (11-22,25). In particular, M2 hasbeen alleged to be inactive (1,2,5,11,12,21,24),appar ently because its in vivo for-mation in humans is lower than that ofMl (1,2,4,6-8). However, it now seems

likely that not only Ml but also M2 hashypoglycemic and insulin -releasing activ-ity, and this may be so even during ther-apeutic conditions. First, the maximumeffective concentration of plasma Gb islower than previou sly assumed; at least inhealthy volunteers, it would correspondto a maximum oral dose of ^3.5 mg(equaling 5 mg of the U.S. Gb formula-tion) (2 8). Second, at least 50% ofagivenGb dose is transformed to Ml and M2(1-4). Third, because Ml and M2 aremore polar than Gb itself, they wouldpossibly be less bound to plasma albu-

Table 3Individual resultant dose per bod y weight of intravenous Ml, intravenous M2,intravenous Gb, and oral Gb

Subjectno .

12345678Mean SD

SexMMMMFFFF

IntravenousM l

47.942.340.746.942.955.761.551.4

48.66 7.20

Dose per body w eight /xg/kg)Intravenous

M243.138.538.150.744.160.556.747.7

47.43 8.14

IntravenousGb

30.936.133.729.828.739.437.242.834.83 4.95

Oral Gb47.745.643.843.646.359.361.154.350.21 7.03

For oral Gb, the nominal potency of the tablet (3.5 mg) was used for calculations.

min, signifying that a greater fraction oftheir total plasma concentration would beavailable to exert an effect.Even if the hypoglycemic contri-butions of Ml and M2 would be less thanassumed from the current data, both me-tabolites may have clinically relevant ef-fects in diabetic patients, particularly inpatients with renal impairment, becauseboth M l and M2 are more polar than Gband would hence be more dependentupon renal function for their excretion.This conclusion is supported by the ob-servation that the risk of long-lasting, se-vere hypoglycemic reactions is increasedin patients with renal impairment (29-

31),even thou gh G b itselfisindependentof renal function for its elimination (25).In conclusion, our data indicate that theGb metabolites are hypoglycemic in hu-mans because of increased insulin secre-tion. The current findings warrant ex-tended studies concerning the clinicalimportance of the two Gb metabolites.AcknowledgmentsThis study was sup-ported in part by grants from The SwedishAcademy of Pharmaceutical Sciences (GunnarHyltens Memory Fund and Elisabeth

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