European Journal of Clinical Pharmacology © by Springer-Verlag 1979
Eur. J. Clin. Pharmacol. 16, 195-202 (1979)
Pharmacokinetics of Mefformin After Intravenous and Oral Administration to Man P. J. Pentik~iinen 1, P. J. Neuvonen 2, and Aneri Penttilfi 3 1SecondDepartment of Medicine and 2Department of Clinical Pharmacology, Universityof Helsinki, and 3ResearchLaboratories of Mediea Ltd., Helsinki, Finland
Summary. The kinetics of 14C-metformin have been studied in five healthy subjects after oral and intravenous administration. The intravenous dose was distributed to a small central compartment of 9.9 + 1.61 (X _+ SE), from which its elimination could be described using three-compartment open model. The elimination half-life from plasma was 1.7 + 0.1 h. Urinary excretion data revealed a quantitatively minor terminal elimination phase with a half-life of 8.9 -_~ 0.7 h. After the intravenous dose, mefformin was completely excreted unchanged in urine with a renal clearance of 454 _+ 47 ml/min. Metformin was not bound to plasma proteins. The concentration of metformin in saliva was considerably lower than in plasma and declined more slowly. The bioavailability of metformin tablets averaged 50--60%. The rate of absorption was slower than that of elimination, which resulted in a plasma concentration profile of "flipflop" type for oral metformin.
8], to prevent incorporation of cholesterol in atherosclerotic lesions [9] and to cause changes in lipoprotein structure and metabolism [10]. Pharmacokinetic information about metformin is scanty. Studies have been hampered by difficulty in the quantitation of metformin in biological samples. On the basis of excretion in urine, the half-life of metformin elimination after oral administration has been reported to be about 3 h [11]. However, Lennard et al. [12], using a sensitive GLC method recently, found evidence of a considerably slower terminal elimination phase in man. Since the toxic effects of metformin have been associated with exceptionally high plasma concentrations of the drug [13], detailed information about its pharmacokinetics would be of importance in planning rational dosing regimens. The present paper describes the pharmacokinetics of metformin absorption and elimination in healthy volunteers.
Key words: metformin, biguanides; pharmacokinetics, absorption
Material and Methods
Metformin (N,N-dimethylbiguanide; Fig. 1) is an oral antidiabetic agent of biguanide type, which has widely replaced phenformin in the treatment of maturity onset diabetes. The recommendation of metformin as the biguanide of choice has been based on the lesser risk of associated lactic acidosis than after use of phenformin [1, 2, 3, 4!1. In addition to its antidiabetic activity, metformin has a hypolipidemic effect, especially in patients with hypertriglyceridemia [5, 6]. It has also been shown to prevent lipid disturbances and atherosclerotic lesions induced by an atherosclerotic diet in experimental animals [7,
Volunteers. Five healthy volunteers participated in the study (Table 1). They had no history of renal, hepatic or gastrointestinal disease and the results of physical examination before the study were normal. The endogenous creatinine clearance of the participants was within the normal range. Informed written consent was obtained from each volunteer before the study.
CH3~N&_HN--.~C--NH2 CHS II II NH NH
. HC I
Fig. 1. Structural formulaof metforminHC1with asterisksshowing the position of 14Clabel
0031-6970/79/0016/0195/$01.60
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P.J. Pentikfiinenet al.: Pharmacokineticsof Metformin
Table 1. Details of the five healthyvolunteersin the pharmacokineticstudy of 14C-mefformin Subject
Age (years)
Sex
Height (era)
Weight (kg)
Serum creatinine (gmol/1)
Creatinine clearance (ml/min/1.73 m2)
K.L. P.P. S.R. T.K. R.L.
36 38 39 46 51
F M F F M
171 171 161 153 186
58 63 60 56 80
71 75 71 80 73
88 111 93 76 136
14C-Metformin. ~4C-Metformin HC1 was prepared by fusion of an equimolar mixture of dimethylamine hydrochloride and 14C-dicyandiamide at 160°C for 2h. The product was purified by preparative chromatography followed by recrystallization from n-propanol. The radiochemical purity was found to exceed 99% by thin-layer chromatography in the solvent systems described below, followed by autoradiography and counting of the radioactivity. Ampoules containing 4.4 ~tCi/500 mg of lgC-metformin HC1 in 10 ml of aqueous solution were prepared. Tablets containing ~4C-metformin HC1 were prepared using the same incredients as in the commercial product (Diformin®, Medica Ltd., Helsinki). Each tablet contained 4.4 ~tCi/500 mg of 14C-metformin HC1. Administration of Drug and Sampling. A dose of 14Cmetformin HC1 4.4 ~tCi/500 mg was given orally or intravenously, in the morning after an overnight fast. All volunteers received the oral dose and three of them were also given the intravenous dose. The doses were given in random order, at least 3 weeks apart. The oral dose was ingested with water 200 ml, and the intravenous dose was injected into a cubital vein over 5min. Blood samples were collected in heparinized tubes at 0, 5, 10, 20, 30, 45 min and 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 and 24 h after the oral dose. In the intravenous experiments, additional samples were taken at the end of the injection and after 2 and 15 min. Urine was collected over: 0-1, 1-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-24, 24-32 and 32--48h after the dose. Faeces were collected throughout one week in all the intravenous experiments and in one of the oral experiments. Saliva sampies, obtained after stimulating saliva flow by chewing waxed film, and samples of expired air [14], were collected 1, 2, 3, 5, 8 and 12 h after administration of the drug. Analysis of 14C-Metformin. The total radioactivity in plasma, saliva, urine and breath samples was measured by liquid scintillation counting and corrected for quenching by internal standardization. Homogenized
samples of faeces were solubilized and decolourized before counting. Since it could be demonstrated that metformin was not metabolized, the measured radioactivity was converted to weight units, which are used in the text.
Metabolism of 14C-Metformin. Urine samples collected after intravenous and oral administration of 14C-metformin, were extracted several times with nbutanol, after making them strongly alkaline with sodium hydroxide. Virtually all radioactivity was extracted by this method. The extract was subjected to two-dimensional thin-layer chromatography using n-butanol-acetic acid-water (60:15:25) and 1 M ammonium acetate: pH 10-ethanol (25:75) as solvent systems. Chromatograms of urine samples with high radioactivity were run directly without preextraction. Autoradiograms were prepared from the chromatograms and the plates were subsequently cut in pieces which were counted in liquid scintillation counter. Protein Binding of 14C-Metformin. Plasma samples obtained 15 min after the intravenous dose, and samples with highest radioactivity after the oral dose of 14C-metformin, were used. In addition, the protein binding of 14C-metformin in vitro was studied in concentrations ranging from 0.05 ~tg/ml to 50 gg/ml. The binding was determined both by equilibrium dialysis [15] and by ultrafiltration [16]. Equilibrium dialysis was performed at 20°C for 13 h against phosphate buffer pH 7.4. Pharmacokinetic calculations Intravenous Administration. The post-injection plasma concentration-time curve showed a three exponential decay in semi-logarithmic graphs. To obtain the corresponding pharmacokinetic parameters the plasma data were fitted to a three-compartment open model [17] based on the equation Cp = A'e - ~ + B'e -~t + C'e -~t
(1)
P. J. Pentikfiinen et al.: Pharmacokinetics of Metformin
197
where Cp is the plasma concentration at the end of the injection and a / 3 and y are the rate constants during the three exponential phases. A', B' and C' represent the contribution of the corresponding exponentials at t = 0 (end of the injection). The initial estimates of these constants were obtained graphically and then subjected to the non-linear least-square regression program Nonlin [18]. The best agreement between the theoretical curve and experimental data points was obtained by use of reciprocal concentration as a weighting factor. Before determining the microscopic rate constants associated with the three-compartment model, the intercepts with the ordinate for an instantenous intravenous bolus injection of the same amount of the drug A, B, and C were calculated from A', B', and C' by the method of Loo and Riegelman [19]. The total plasma clearance was calculated by the equation Dose Plasma clearance - - - kel X Vd c (2) AUC where AUC~ is the area under the plasma concentration time curve from the start of the injection including the infinite area. AUC until 10 or 12 h was calculated by the trapezoidal rule and was corrected for infinity using the rate constant of the last exponential phase. The volume of the central compartment (Vdc) was obtained according to Equation 2 by dividing the plasma clearance by the elimination rate constant from the central compartment (k,.@ The apparent volume at pseudodistribution e q u i l i b r i u m (Vdarea) was calculated according to equation Dose Vd,~e~ (3) AUC" y Renal clearance was obtained as the slope of the regression line of the plot of excretion rate versus plasma concentration at the midpoint of the urine collection period. Oral Administration. The AUC after oral administration of 14C-metformin was calculated as in the intravenous experiments. The plasma concentration time curve was described according to equation Cp = Ae -a't -- Be -#'t
(4)
where a' is the rate constant of the terminal linear portion of the curve and/3' the corresponding constant for first part of the curve. Paradoxically, a' corresponds to the absorption rate constant of metformin according to the "flip-flop"-model [20], i. e., the ln2 absorption half-life is - 0(
The renal clearance of me~:formin after oral administration was calculated as in the intravenous experiments.
I
front
l c
>= o
14.
~N X
:0
Second solvent
.=
!
Fig. 2. Autoradiogram of a two-dimensional thin-layer chromato~ a m of urine (40 gl) collected between 4 and 6 hours after intravenous administration of 4.4 gCi/500 mg I4C-metformin HC1 to a healthy volunteer. The first solvent was 1 M ammonium acetate pH 10-ethanol (25 : 75) and the second solvent was n-butanolacetic acid-water (60: 15 : 25)
Metabolic Fate of z4C-Metformin Chromatograms of urine, run directly or after extraction with n-butanol, showed only one radioactive compound. This had identical Rf values to standard metformin chromatographed on silica gel plates in the solvent systems n-butanol-acetic acid-water (Rf 0.35), and in 1 M ammonium acetate-ethanol (Rf 0.50). A demonstration autoradiogram is presented in Figure 2. Counting of the thin-layer plates confirmed the presence of only one radioactive compound.
Results
Intravenous Administration Plasma Data. Plasma concentration could be measured for up to 10-12 h after administration of 14Cmetformin. Observed concentrations and computer fitted curves for each volunteer are shown in Figure 3, which also demonstrates the good fit obtained with a three-compartment model. The parameters of the model are listed in Table 2 and some model-independent variables of metformin kinetics are given in Table 3. Metformin was distributed to a small central compartment with a mean volume of 9.9 + 1.61 (X + SE). The first two rapid phases mainly represent dis-
198
P. J. Pentik~iinen et al.: Pharmacokinetics of Metformin 20 10
--~ E
10
10
0.1
0.1
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"~ O~ Z.
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0.5
Eur. J. Clin. Pharmacol. 16, 195-202 (1979)
Pharmacokinetics of Mefformin After Intravenous and Oral Administration to Man P. J. Pentik~iinen 1, P. J. Neuvonen 2, and Aneri Penttilfi 3 1SecondDepartment of Medicine and 2Department of Clinical Pharmacology, Universityof Helsinki, and 3ResearchLaboratories of Mediea Ltd., Helsinki, Finland
Summary. The kinetics of 14C-metformin have been studied in five healthy subjects after oral and intravenous administration. The intravenous dose was distributed to a small central compartment of 9.9 + 1.61 (X _+ SE), from which its elimination could be described using three-compartment open model. The elimination half-life from plasma was 1.7 + 0.1 h. Urinary excretion data revealed a quantitatively minor terminal elimination phase with a half-life of 8.9 -_~ 0.7 h. After the intravenous dose, mefformin was completely excreted unchanged in urine with a renal clearance of 454 _+ 47 ml/min. Metformin was not bound to plasma proteins. The concentration of metformin in saliva was considerably lower than in plasma and declined more slowly. The bioavailability of metformin tablets averaged 50--60%. The rate of absorption was slower than that of elimination, which resulted in a plasma concentration profile of "flipflop" type for oral metformin.
8], to prevent incorporation of cholesterol in atherosclerotic lesions [9] and to cause changes in lipoprotein structure and metabolism [10]. Pharmacokinetic information about metformin is scanty. Studies have been hampered by difficulty in the quantitation of metformin in biological samples. On the basis of excretion in urine, the half-life of metformin elimination after oral administration has been reported to be about 3 h [11]. However, Lennard et al. [12], using a sensitive GLC method recently, found evidence of a considerably slower terminal elimination phase in man. Since the toxic effects of metformin have been associated with exceptionally high plasma concentrations of the drug [13], detailed information about its pharmacokinetics would be of importance in planning rational dosing regimens. The present paper describes the pharmacokinetics of metformin absorption and elimination in healthy volunteers.
Key words: metformin, biguanides; pharmacokinetics, absorption
Material and Methods
Metformin (N,N-dimethylbiguanide; Fig. 1) is an oral antidiabetic agent of biguanide type, which has widely replaced phenformin in the treatment of maturity onset diabetes. The recommendation of metformin as the biguanide of choice has been based on the lesser risk of associated lactic acidosis than after use of phenformin [1, 2, 3, 4!1. In addition to its antidiabetic activity, metformin has a hypolipidemic effect, especially in patients with hypertriglyceridemia [5, 6]. It has also been shown to prevent lipid disturbances and atherosclerotic lesions induced by an atherosclerotic diet in experimental animals [7,
Volunteers. Five healthy volunteers participated in the study (Table 1). They had no history of renal, hepatic or gastrointestinal disease and the results of physical examination before the study were normal. The endogenous creatinine clearance of the participants was within the normal range. Informed written consent was obtained from each volunteer before the study.
CH3~N&_HN--.~C--NH2 CHS II II NH NH
. HC I
Fig. 1. Structural formulaof metforminHC1with asterisksshowing the position of 14Clabel
0031-6970/79/0016/0195/$01.60
196
P.J. Pentikfiinenet al.: Pharmacokineticsof Metformin
Table 1. Details of the five healthyvolunteersin the pharmacokineticstudy of 14C-mefformin Subject
Age (years)
Sex
Height (era)
Weight (kg)
Serum creatinine (gmol/1)
Creatinine clearance (ml/min/1.73 m2)
K.L. P.P. S.R. T.K. R.L.
36 38 39 46 51
F M F F M
171 171 161 153 186
58 63 60 56 80
71 75 71 80 73
88 111 93 76 136
14C-Metformin. ~4C-Metformin HC1 was prepared by fusion of an equimolar mixture of dimethylamine hydrochloride and 14C-dicyandiamide at 160°C for 2h. The product was purified by preparative chromatography followed by recrystallization from n-propanol. The radiochemical purity was found to exceed 99% by thin-layer chromatography in the solvent systems described below, followed by autoradiography and counting of the radioactivity. Ampoules containing 4.4 ~tCi/500 mg of lgC-metformin HC1 in 10 ml of aqueous solution were prepared. Tablets containing ~4C-metformin HC1 were prepared using the same incredients as in the commercial product (Diformin®, Medica Ltd., Helsinki). Each tablet contained 4.4 ~tCi/500 mg of 14C-metformin HC1. Administration of Drug and Sampling. A dose of 14Cmetformin HC1 4.4 ~tCi/500 mg was given orally or intravenously, in the morning after an overnight fast. All volunteers received the oral dose and three of them were also given the intravenous dose. The doses were given in random order, at least 3 weeks apart. The oral dose was ingested with water 200 ml, and the intravenous dose was injected into a cubital vein over 5min. Blood samples were collected in heparinized tubes at 0, 5, 10, 20, 30, 45 min and 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 and 24 h after the oral dose. In the intravenous experiments, additional samples were taken at the end of the injection and after 2 and 15 min. Urine was collected over: 0-1, 1-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-24, 24-32 and 32--48h after the dose. Faeces were collected throughout one week in all the intravenous experiments and in one of the oral experiments. Saliva sampies, obtained after stimulating saliva flow by chewing waxed film, and samples of expired air [14], were collected 1, 2, 3, 5, 8 and 12 h after administration of the drug. Analysis of 14C-Metformin. The total radioactivity in plasma, saliva, urine and breath samples was measured by liquid scintillation counting and corrected for quenching by internal standardization. Homogenized
samples of faeces were solubilized and decolourized before counting. Since it could be demonstrated that metformin was not metabolized, the measured radioactivity was converted to weight units, which are used in the text.
Metabolism of 14C-Metformin. Urine samples collected after intravenous and oral administration of 14C-metformin, were extracted several times with nbutanol, after making them strongly alkaline with sodium hydroxide. Virtually all radioactivity was extracted by this method. The extract was subjected to two-dimensional thin-layer chromatography using n-butanol-acetic acid-water (60:15:25) and 1 M ammonium acetate: pH 10-ethanol (25:75) as solvent systems. Chromatograms of urine samples with high radioactivity were run directly without preextraction. Autoradiograms were prepared from the chromatograms and the plates were subsequently cut in pieces which were counted in liquid scintillation counter. Protein Binding of 14C-Metformin. Plasma samples obtained 15 min after the intravenous dose, and samples with highest radioactivity after the oral dose of 14C-metformin, were used. In addition, the protein binding of 14C-metformin in vitro was studied in concentrations ranging from 0.05 ~tg/ml to 50 gg/ml. The binding was determined both by equilibrium dialysis [15] and by ultrafiltration [16]. Equilibrium dialysis was performed at 20°C for 13 h against phosphate buffer pH 7.4. Pharmacokinetic calculations Intravenous Administration. The post-injection plasma concentration-time curve showed a three exponential decay in semi-logarithmic graphs. To obtain the corresponding pharmacokinetic parameters the plasma data were fitted to a three-compartment open model [17] based on the equation Cp = A'e - ~ + B'e -~t + C'e -~t
(1)
P. J. Pentikfiinen et al.: Pharmacokinetics of Metformin
197
where Cp is the plasma concentration at the end of the injection and a / 3 and y are the rate constants during the three exponential phases. A', B' and C' represent the contribution of the corresponding exponentials at t = 0 (end of the injection). The initial estimates of these constants were obtained graphically and then subjected to the non-linear least-square regression program Nonlin [18]. The best agreement between the theoretical curve and experimental data points was obtained by use of reciprocal concentration as a weighting factor. Before determining the microscopic rate constants associated with the three-compartment model, the intercepts with the ordinate for an instantenous intravenous bolus injection of the same amount of the drug A, B, and C were calculated from A', B', and C' by the method of Loo and Riegelman [19]. The total plasma clearance was calculated by the equation Dose Plasma clearance - - - kel X Vd c (2) AUC where AUC~ is the area under the plasma concentration time curve from the start of the injection including the infinite area. AUC until 10 or 12 h was calculated by the trapezoidal rule and was corrected for infinity using the rate constant of the last exponential phase. The volume of the central compartment (Vdc) was obtained according to Equation 2 by dividing the plasma clearance by the elimination rate constant from the central compartment (k,.@ The apparent volume at pseudodistribution e q u i l i b r i u m (Vdarea) was calculated according to equation Dose Vd,~e~ (3) AUC" y Renal clearance was obtained as the slope of the regression line of the plot of excretion rate versus plasma concentration at the midpoint of the urine collection period. Oral Administration. The AUC after oral administration of 14C-metformin was calculated as in the intravenous experiments. The plasma concentration time curve was described according to equation Cp = Ae -a't -- Be -#'t
(4)
where a' is the rate constant of the terminal linear portion of the curve and/3' the corresponding constant for first part of the curve. Paradoxically, a' corresponds to the absorption rate constant of metformin according to the "flip-flop"-model [20], i. e., the ln2 absorption half-life is - 0(
The renal clearance of me~:formin after oral administration was calculated as in the intravenous experiments.
I
front
l c
>= o
14.
~N X
:0
Second solvent
.=
!
Fig. 2. Autoradiogram of a two-dimensional thin-layer chromato~ a m of urine (40 gl) collected between 4 and 6 hours after intravenous administration of 4.4 gCi/500 mg I4C-metformin HC1 to a healthy volunteer. The first solvent was 1 M ammonium acetate pH 10-ethanol (25 : 75) and the second solvent was n-butanolacetic acid-water (60: 15 : 25)
Metabolic Fate of z4C-Metformin Chromatograms of urine, run directly or after extraction with n-butanol, showed only one radioactive compound. This had identical Rf values to standard metformin chromatographed on silica gel plates in the solvent systems n-butanol-acetic acid-water (Rf 0.35), and in 1 M ammonium acetate-ethanol (Rf 0.50). A demonstration autoradiogram is presented in Figure 2. Counting of the thin-layer plates confirmed the presence of only one radioactive compound.
Results
Intravenous Administration Plasma Data. Plasma concentration could be measured for up to 10-12 h after administration of 14Cmetformin. Observed concentrations and computer fitted curves for each volunteer are shown in Figure 3, which also demonstrates the good fit obtained with a three-compartment model. The parameters of the model are listed in Table 2 and some model-independent variables of metformin kinetics are given in Table 3. Metformin was distributed to a small central compartment with a mean volume of 9.9 + 1.61 (X + SE). The first two rapid phases mainly represent dis-
198
P. J. Pentik~iinen et al.: Pharmacokinetics of Metformin 20 10
--~ E
10
10
0.1
0.1
5 2
"~ O~ Z.
1
0.5
