Intersex and Interspecies Pharmacokinetics of Metoprolol After Oral and Intravenous Dose Administration in Rats and Mice

Abstract

Aim: Intersex and interspecies metoprolol pharmacokinetics following intravenous and oral dose administration in rodents. Materials & methods: Oral and intravenous dose studies were conducted in rats and mice. Significant intersex differences were observed in peak plasma levels of metoprolol after oral dose in both the species. The plasma concentration (C_max) was approximately sevenfold higher (270.356 ng/ml) in female compared with male rats (40.981 ng/ml) following oral dose administration. The C_max observed for male (878.822 ± 75.5 ng/ml) was approximately twofold higher than in female mouse (404.016 ± 113.5 ng/ml) after oral dose administration. Conclusion: Sex and species related physioanatomical characteristics alters metoprolol pharmacokinetics. Such differences should be addressed in studies related to metoprolol interactions with concurrently administered drug candidates.

Graphical abstract

Keywords::

• bioavailability
• drug interaction
• half-life
• systemic exposure

In the recent years, the advancement in the pharmacological research has explored and augmented our fundamental understandings underlying the variabilities in pharmacokinetics (PK) of drugs. There are huge number of variables that affect the PK of drug in between the sexes and among different species. Besides the intersex and interspecies differences in the physiochemistry, individual genetic dispositions have been found to exert substantial differences in the PK. Sex related factors have been reported to significantly influence the metabolism of drugs like metoprolol, propranolol etc. [1,2]. During the early stages of drug discovery and development, the PK properties of drug candidates are explored along with routine analysis of drug interaction potential. Such studies are imperative to assess the safety profile of drug candidates. Preclinical studies in experimental animals are often employed to screen the interaction potential of drug candidates [3,4]. The outcomes of preclinical studies can be used to predict human PK using allometric scaling approaches. This approach considers important physiological and anatomical variables in higher species as a power function of the body weights across the species to predict human PK [5]. While evaluating the interaction potential of drugs in preclinical species, considering the intersex and interspecies influences on PK could effectively estimate the need of interaction studies in clinical phase.

Cardiovascular disease is one among the most prevalent killer disease, responsible for one in every three deaths globally. Metoprolol is a selective β1-adrenergic receptor blocker clinically used to treat many cardiovascular disease including hypertension, angina pectoris, arrhythmia, myocardial infarction and other cardiovascular diseases. The Metoprolol Atherosclerosis Prevention in Hypertensives (MAPHY) trials have shown a benefit of metoprolol over diuretics regarding sudden cardiac death as well as myocardial infarction. A large, randomized trial of over 50,000 patients in the 1990s showed metoprolol to reduce mortality and reinfarction when used chronically after myocardial infarction [6]. The metoprolol plasma concentration and the β1 receptor antagonist activity have good correlation in-vivo. It shows complete absorption from GI tract (GIT) following oral dose administration. It exhibits low bioavailabilities due to significant hepatic first-pass biotransformation and is predominantly metabolized by cytochrome P-450 2D6 (CYP2D6) [7]. The present study was conducted to investigate the intersex and interspecies differences in the PK of metoprolol following single dose intravenous and oral administration of metoprolol tartrate in male and female rat and mouse.

Experimental

Chemicals & reagents

Acetonitrile and methanol of HPLC grade were purchased from Merck (Darmstadt, Germany). Formic acid of HPLC grade was obtained from ROE (NJ, USA). Metoprolol tartrate (purity >98%) and internal standard (IS) telmisartan were procured from Sigma Aldrich, MO, USA. All other chemicals and reagents were of analytical LC grade. Drug-free rat and mouse plasma was collected from healthy Sprague–Dawley (SD) rats and CD1 mice obtained from in-house animal facility. All animal experimentation and procedures were done as per the protocols approved by Institutional Animal Ethics Committee (IAEC, Approval number: ARAGENB-IAEC-0001-01-22).

Subjects

Healthy adult SD rats (200–250 g) and CD-1 mice (25–35 g) of both the sexes were acclimatized for 3 days prior to the study in proper ventilated polypropylene cases under controlled standard laboratory conditions of regular 12 h light–dark cycle, temperature (22 ± 2°C) and relative humidity (55 ± 5%). Certified rodent diet and water was provided to ad libitum. Animals were kept for overnight fasting prior to studies. Animals were maintained and monitored for good health in accordance with test facility standard operating procedures. Guidelines approved by the GLP were followed throughout the animal experimentation.

Study design

The studies were parallel single dose oral and intravenous plasma PK, designed to estimate the intersex and interspecies influences on the PK profile of metoprolol after administration of metoprolol tartrate. The studies were conducted in four groups of experimental animals for each species. Every group of animals comprised three animals (n = 3). Studies were conducted in discrete group of sexes in both the species. Since this is an exploratory preclinical study and further no significant inter-animal variations were observed in the PK profile, three animals per group were considered in the study. In the preliminary studies conducted on rats (data not shown in this article), we did not find significant inter-animal variations in PK, based on this n = 3 was considered for this study. Metoprolol doses were selected in reference to various nonclinical PK exploratory studies conducted in rat and mouse [7,8]. Rats of group 1 (male) and 2 (female) were dosed intravenous at 1 mg/kg and group 3 (male) and 4 (female) were dosed orally at 5 mg/kg of metoprolol. Similarly, mice of group 1 (male) and 2 (female) were dosed intravenously at 2 mg/kg and group 3 (male) and 4 (female) were dosed orally at 10 mg/kg of metoprolol. Blood samples were collected at 0.033, 0.25, 0.5, 1, 2, 4, 8, 24 h post-dose following intravenous dose and 0.033, 0.333, 1, 2, 4, 8, 24 h post-dose following oral dose administration in rats. For mouse studies, blood samples were collected at 0.033, 0.333, 1, 2, 4, 8, 24 h post-dose following intravenous dose and 0.033, 0.333, 1, 2, 4, 8, 24 h post-dose following oral dose administration. Blood samples were collected through jugular vein in rats and saphenous vein in mice. The rats were surgically operated for jugular vein cannulation 3 days before the commencement of study and were closely observed throughout the recovery period.

Formulation

Fresh metoprolol tartrate formulations were prepared in normal saline (0.85% NaCl in water) for both intravenous and oral dose on the day of dosing. The volume factor was 5 ml/kg for intravenous dose and 10 ml/kg for oral dose in both the species.

Bioanalysis

Blood samples collected from the respective studies were immediately centrifuged to obtain plasma. The plasma samples were stored at -70°C till analysis. Before analysis, sample were thawed at room temperature and 10 μl sample was aliquoted for further processing. Samples was crashed using 150 μl acetonitrile containing telmisartan (IS; 20 ng/ml) and vortexed for about 2 min. Resulting mixture was centrifuged at 4000 r.p.m. for 7 min at 4°C. From the supernatant, 100 μl volume was separated and diluted with 100 μl Milli-Q water. This solution was transferred in to HPLC vails and subsequently subjected to LC–MS/MS analysis. Plasma concentration was determined by using a partially validated LC–MS/MS method. Mass spectrometric detection was performed on API 6500 LC–MS/MS mass spectrometer (Applied Bio systems, Sciex, MA, USA) with Analyst 1.7 software. Parent to product ion transitions at m/z 268.20 to 116.20 and 515.20 to 276.20 were used for quantification of metoprolol and IS respectively. The assay was linear over the range 1.08–5000 ng/ml with lower limit of quantification of 1.08 ng/ml. The concentrations at individual calibration levels were 1.08, 2.02, 10.80, 60.0, 300.0, 1200, 2400, 4000 and 5000 ng/ml. Coefficients of determination (r²) were >0.990 for standard curves generated. Precision and accuracy of the method was determined by analyzing quality control samples at 6.05, 2520 and 4200 ng/ml.

PK & statistical analysis

The primary end points for these studies were area under the curve (AUC), maximum plasma concentration (C_max and C₀), time to attain C_max (T_max), volume of distribution (Vd), clearance (CL), elimination half-life (T_1/2), mean residence time and absolute bioavailability (%F). The PK parameters were calculated by non compartmental analysis using Winnonlin Phoenix (Version 8.1, Pharsight Corporation). The PK parameters were statistically compared using two-tailed Student’s t-test for analyzing in intersex and interspecies variability. In all the tests, a probability level of significance was kept at α = 0.05. Results were expressed as mean ± SD; n = 3). The bioavailability was calculated as:$\mathrm{Bioavilability}=\frac{AUC\mathrm{oral}}{AUCIV}\times \frac{\mathrm{Dose}IV}{\mathrm{Dose}\mathrm{oral}}$

Results

Metoprolol PK in SD rats

Metoprolol tartrate intravenous administration in male & female SD rats

Table 1 summarizes calculated PK parameters of metoprolol following intravenous dose administration of metoprolol tartrate in male and female SD rats. The mean plasma concentration–time profile is shown in Figure 1.

Table 1. Pharmacokinetic parameters of metoprolol following intravenous dose administration of metoprolol tartrate in Sprague–Dawley rat (n=3; mean ± standard deviation) at 1.0 mg/kg dose.

PK parameters	Metoprolol (iv.; 1 mg/kg of metoprolol tartrate, n = 3)
	Male SD rats	Female SD rats
C0 (ng/ml)	455.345 ± 125.2	463.007 ± 168.3
T1/2 (h)	0.65 ± 0.2	1.03 ± 0.3
Vd (l/kg)	4.215 ± 1.3	8.125 ± 2.4
Cl (ml/min/kg)	76.42 ± 6.4	92.76 ± 11.2
AUClast (h × ng/ml)	215 ± 22.6	180 ± 23.2
AUCinf. (h × ng/ml)	219 ± 18.8	182 ± 23.2
MRTlast (h)	0.67 ± 0.2	0.80 ± 0.2

AUC: Area under the curve; C₀: Plasma drug concentration at T=0; Cl: Plasma drug clearance; iv.: Intravenous; MRT: Mean residence time; PK: Pharmacokinetic; SD: Sprague–Dawley; T_1/2: Elimination half-life; Vd: Volume of distribution.

Figure 1. Concentration–time profile of metoprolol after intravenous dose administration of metoprolol tartrate in male and female rat (n = 3) at 1 mg/kg dose.

mpk: mg/kg.

Metoprolol tartrate oral administration in male & female SD rats

Table 2 summarizes calculated PK parameters of metoprolol following oral dose administration of metoprolol tartrate in male and female SD rats. The mean plasma concentration–time profile is shown in Figure 2.

Table 2. Pharmacokinetic parameters of metoprolol following oral dose administration of metoprolol tartrate in SD rat (n=3; mean ± standard deviation) at 5.0 mg/kg dose.

PK parameters	Metoprolol (oral; 1 mg/kg of metoprolol tartrate, n = 3)
	Male SD rats	Female SD rats
Cmax (ng/ml)	40.981 ± 7.7	270.356 ± 25.4
T1/2 (h)	3.35 ± 0.6	1.342 ± 0.2
Vd_F (lkg)	325.414 ± 78.5	40.479 ± 1.9
ClF (ml/min/kg)	1151.65 ± 332.7	350.07 ± 32.9
AUClast (h × ng/ml)	175 ± 7.9	236 ± 22.3
AUCinf. (h × ng/ml)	NRV ^†	240 ± 23.1
MRTlast (h)	2.00 ± 0.7	1.12 ± 0.2

† NRV: Not reportable value (% extrapolated of total AUC≥120%AUC_0-last).

AUC: Area under the curve; C_max: Maximum plasma drug concentration; Cl_F: Apparent oral clearance; MRT: Mean residence time; PK: Pharmacokinetic; SD: Sprague Dawley; T_1/2: Elimination half-life; Vd_F: Apparent volume of distribution.

Figure 2. Concentration–time profile of metoprolol after oral dose administration of metoprolol tartrate in male and female rat (n = 3) 5 mg/kg dose.

Metoprolol PK in CD1 mouse

Metoprolol tartrate intravenous administration in male & female CD1 mouse

Table 3 summarizes calculated PK parameters of metoprolol following intravenous dose administration of metoprolol tartrate in male and female mouse. The mean plasma concentration–time profile is shown in Figure 3.

Table 3. Pharmacokinetic parameters of metoprolol following intravenous dose administration of metoprolol tartrate in CD1 mouse (n=3; mean ± standard deviation) at 2.0 mg/kg dose.

PK parameters	Metoprolol (iv.; 2 mg/kg of metoprolol tartrate, n = 3)
	Male CD1 mouse	Female CD1 mouse
C0 (ng/ml)	1144.025 ± 72.3	775.926 ± 154.4
T1/2 (h)	1.13 ± 0.5	1.31 ± 0.5
Vd (l/kg)	3.97 ± 0.8	15.103 ± 4.7
Cl (mL/min/kg)	113.26 ± 12.6	138.42 ± 21.0
AUClast (h × ng/ml)	293 ± 34.6	241 ± 35.2
AUCinf. (h × ng/ml)	297 ± 35.1	244 ± 35.7
MRTlast (h)	0.49 ± 0.1	0.61 ± 0.1

AUC: Area under the curve; C0: Plasma drug concentration at T=0; Cl: Plasma drug clearance; iv.: Intravenous; MRT: Mean residence time; PK: Pharmacokinetic; SD: Sprague–Dawley; T1/2: Elimination half-life; Vd: Volume of distribution.

Figure 3. Concentration–time profile of metoprolol after intravenous dose administration of metoprolol tartrate in male and female mouse (n = 3) at 2 mg/kg dose.

Metoprolol tartrate oral administration in male & female CD1 mouse

Table 4 summarizes calculated PK parameters of metoprolol following oral dose administration of metoprolol tartrate in male and female mouse. The mean plasma concentration–time profile is shown in Figure 4.

Table 4. Pharmacokinetic parameters of metoprolol following oral dose administration of metoprolol tartrate in CD1 mouse (n=3; mean ± SD) at 10.0 mg/kg dose.

PK parameters	Metoprolol (oral; 10 mg/kg of metoprolol tartrate, n = 3)
	Male CD1 mouse	Female CD1 mouse
Cmax (ng/ml)	878.822 ± 75.5	404.016 ± 113.5
T1/2 (h)	1.55 ± 1.3	1.07 ± 1.0
Vd_F (l/kg)	34.36 ± 22.6	49.13 ± 57.2
ClF (ml/min/kg)	280.60 ± 68.1	457.37 ± 132.9
AUClast (h × ng/ml)	602 ± 133.4	380 ± 118.6
AUCinf. (h × ng/ml)	619 ± 153.9	388 ± 120.8
MRTlast (h)	0.93 ± 0.4	1.17 ± 0.2

AUC: Area under the curve; C0: Plasma drug concentration at T=0; Cl: Plasma drug clearance; iv.: Intravenous; MRT: Mean residence time; PK: Pharmacokinetic; SD: Sprague–Dawley; T1/2: Elimination half-life; Vd: Volume of distribution.

Figure 4. Concentration–time profile of metoprolol after oral dose administration of metoprolol tartrate in male and female mouse (n = 3) at 10 mg/kg dose.

Discussion

The variability in the PK of drugs among various species is well established and understood, which is attributed to the differences in the physiology of organisms. Besides this, there are several sex-dependent and sex-specific physio-anatomical characteristics contributing to the variations in the PK. The differences in the PK of a drug due to intersex influences could potentially require some dose adjustment strategies to achieve desired pharmacology. While pharmacokinetic differences due to interspecies influences help the researchers to identify a suitable preclinical species to generate relevant data for conducting clinical experiments. Therefore, it is important to explore the existence of such differences in the PK of drugs [9–11]. The present study was conducted to explore and evaluate intersex and interspecies differences in the PK of metoprolol following single oral and intravenous dose administration of metoprolol tartrate in two rodent species viz rat and mouse. However, the results of this study are indicative of the existence of the intersex and interspecies differences in metoprolol PK. Such explorative results may indicate the need to assess and estimate the existence of such differences while studying pharmacokinetic-pharmacodynamics of metoprolol.

Metoprolol exhibited similar PK following intravenous bolus administration of metoprolol tartrate in male and female rats. The values of AUC_0-last were 215 ± 22.6 ng × h/ml and 180 ± 23.2 ng × h/ml in male and female rats respectively. The corresponding C₀ values were 455.345 ± 125.2 ng/ml and 463.007 ± 168.3 ng/ml (Figure 1). In both the sexes, the fraction of AUC extrapolated to infinity (AUC_0-inf.) was within 20% of total AUC values indicating that elimination was monoexponentially linear, and no drug remained in the body after last sample collection. No statistically significant differences were obtained in other pharmacokinetic parameters, such as CL and Vd in terms of p < 0.05 (Table 1).

However, similar pattern was not observed in PK of metoprolol following single oral dose administration of metoprolol tartrate in rats. Statistically significant differences (p < 0.05) were obtained in all the pharmacokinetic parameters except T_1/2 and mean residence time values. The average peak plasma concentration (C_max) was approximately sevenfold higher (270.356 ng/ml) in female rats compared with (40.981 ng/ml) in male rats (Figure 2). The time to reach C_max (T_max) range was 0.25 h to 0.5 h post-dose in female rats, while T_max was 0.25 h post-dose for male rats. Corresponding statistically significant differences were also observed in all related pharmacokinetic parameters (Table 2). Overall, the female rats exhibited significantly different PK compared with male rats after oral dose administration of metoprolol tartrate.

The distinctions in the metoprolol PK between male and female rats can be explained based on the intersex differences in various physiological aspects. It is well established that the peak plasma concentration and total exposure of a drug are dependent on the Vd and CL. For most of the drugs Vd and CL are body weight dependent [12]. Although, in our experiment’s metoprolol was dosed based on the animal weight, the differences in the metoprolol pharmacokinetic could not be explained on this basis of related attributes. Once the drugs are given orally, there are number of other confounding covariates that may significantly alter PK between the sexes. It is well known that GIT factors including gastric secretions, gastric emptying rate, GIT blood flow, pre systemic biotransformation, differential transporter expression etc. significantly influences the drug absorption. The differences in the level of hormones are found to modify theses physiological factors [13,14]. It has been found that pre systemic metabolism both in the GIT and liver was one of the responsive factors contributing to the intersex and interspecies differences in the Verapamil PK after oral dose administration [15]. The intersex and interspecies differences in the expression and activity of various enzymatic pathways could be the potential reason for the intersex differences obtained in metoprolol PK following oral dose administration. It is reported that metoprolol is primarily metabolized by CYP2D6. Luzier et. al. found that the levels of metoprolol were significantly higher in female subjects, indicated higher metabolism in men [16,17]. Our finding in animals was like their findings. It was found that the total clearance in male rats was approximately threefold higher (1151.65 ± 332.7 ml/min/kg) compared with female rats (350.07 ± 32.9 ml/min/kg). Corresponding differences were also observed in related derived parameters like AUC_last (~1.5-folds; 236 ± 22.3 and 175 ± 7.9 h × ng/ml) and T_1/2 values (p < 0.05) Table 2.

Unlike in rats, the metoprolol PK was comparatively similar in both male and female mouse following intravenous and oral dose administration of metoprolol tartrate. However, statistically significant differences were observed in peak plasma concentration of metoprolol following both the routes of administration. The average peak metoprolol levels in male mouse (1144.025 ± 72.3 ng/ml) were approximately 1.5-times higher than in female mouse (775.926 ± 154.4 ng/ml) after intravenous dose administration Table 3. Similarly, the C_max observed for male mouse (878.822 ± 75.5 ng/ml) was approximately twofold higher than in female mouse (404.016 ± 113.5 ng/ml) after oral dose administration Table 4. The C_max was achieved at 0.33 h post-dose in both the sexes. The CL values were comparable in both the sexes after intravenous dose administration, but after oral dose administration CL was found to be 1.6-times higher in female mouse (457.37 ± 132.9 ml/min/kg) compared with male mouse 280.60 ± 68.1 ml/min/kg) Tables 3 & 4. This might be the possible reason for lower peak plasma concentration of metoprolol after oral dose administration in female mouse. Although, the CL values were comparable in both the sexes post-intravenous dose administration, the lower systemic exposure of metoprolol could be in part attributed to higher Vd (15.103 ± 4.7 l/kg) in female mouse compared with lower Vd (3.97 ± 0.8 l/kg) in male mouse Table 3.

Overall, in both the rodent species, in other words, rat and mouse, metoprolol PK was comparable. In both the species, significant differences were observed in the pharmacokinetic parameters of metoprolol specifically after oral dose administration of metoprolol tartrate. Significant intersex differences were observed in peak plasma levels and subsequent systemic exposure of metoprolol in both the species. The mean absolute bioavailabilities were found to be approximately 15% in male rats and 27% in female rats. While in male and female mouse, the values of mean absolute bioavailabilities were approximately 41 and 52% respectively. Figures 5 & 6 represents the relative exposures of metoprolol after intravenous and oral dose administration of metoprolol tartrate in both the sexes of mouse and rat.

Figure 5. AUC_0-t (systemic exposure) of metoprolol after oral and intravenous dose administration of metoprolol tartrate in male and female rat (n = 3).

AUC: Area under the curve.

Figure 6. AUC_0-t (systemic exposure) of metoprolol after oral and intravenous dose administration of metoprolol tartrate in male and female mouse (n = 3).

AUC: Area under the curve.

Conclusion

The results of this study indicated that the sex and species related physiological and anatomical characteristics potentially alters the metoprolol PK irrespective of the route of administration. The results of this study revealed that the metoprolol PK was significant different in male and female animals of both the species. However, the metoprolol PK was similar in between both the species. Such differences should be considered while exploring PK pharmacodynamic studies related to metoprolol interactions with concurrently administered drug candidates.

Limitations

Commercial preparation was not available in this study. This is expected to make differences in a few of the absorption related parameters. However, it would not influence the primary PK parameters of drug like Vd and CL. Overall, the intersex differences if observed will remain same though the magnitude may vary. Since this is an exploratory preclinical study and no significant inter-animal variations were observed in the PK profile, three animals per group were considered in the study. In the preliminary studies conducted on rats (data not shown), we did not find significant inter-animal variations in PK, based on this n = 3 was considered for this study.

Summary points

• Parallel single-dose oral and intravenous studies were conducted to examine intersex and interspecies influences on the pharmacokinetics (PK) profile of metoprolol after administration of metoprolol tartrate.

• Plasma concentration was determined by using a partially validated LC–MS/MS method. Product ion transitions at m/z 268.20–116.20 and 515.20–276.20 were used for quantification of metoprolol and internal standard, respectively.

• Metoprolol exhibited similar PK following intravenous dose administration, while exhibited discrete PK following oral dose administration of metoprolol tartrate in male and female rats.

• Unlike in rats, the metoprolol PK was comparatively similar in both male and female mouse following intravenous and oral dose administration of metoprolol tartrate.

• The mean absolute bioavailabilities were found to be approximately 15% in male rats and 27% in female rats. While in male and female mouse, the values of mean absolute bio availabilities were approximately 41 and 52% respectively.

• It was found that the total clearance in male rats was approximately threefold higher (1151.65 ± 332.7 ml/min/kg) compared with female rats (350.07 ± 32.9 ml/min/kg). Corresponding differences were also observed in related derived parameters like AUC_last (~1.5-folds; 236 ± 22.3 and 175 ± 7.9 h × ng/ml) and T_1/2 values (p < 0.05).

• The CL values for mouse were comparable in both the sexes after intravenous dose administration, but after oral dose administration CL was found to be 1.6-times higher in female mouse (457.37 ± 132.9 ml/min/kg) compared with male mouse 280.60 ± 68.1 ml/min/kg).

• The sex and species related physiological and anatomical characteristics potentially alters the metoprolol PK irrespective of the route of administration. The results of this study revealed that the metoprolol PK were significant different in male and female animals of both the species. However, the metoprolol PK was more or less similar in between both the species.

Author contributions

Y Singh, P Srivastava, and SK Tiwari conceptualized, analyzed the raw data. Y Singh wrote and reviewed the manuscript. D Sahu, T Thykandy, G Bilagi, P Hingole and D Rana conducted the experiments. R AkkiReddy and R Naraganti contributed to the bioanalysis of study samples. Further, the manuscript has been read and approved by all the authors and each author believes that the manuscript represents original work.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.

Acknowledgments

The authors acknowledge and sincerely thank Aragen Life Sciences Private Ltd for providing infrastructure and resources for conducting this experiment. The authors would like to thank S Atole and R Gotte for their consistent technical support throughout the study.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.