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CARDIOVASCULAR

The T- and L-Type Calcium Channel Blocker (CCB) Mibefradil Attenuates Leg Edema Induced by the L-Type CCB Nifedipine in the Spontaneously Hypertensive Rat: A Novel Differentiating Assay

Cardiovascular and Atherosclerosis Biology (T.C.M., M.C.K.), Center of Emphasis-Bioimaging (S.D., N.B., S.L.), Worldwide Comparative Medicine (B.M.), World Wide Safety Sciences (G.S., C.O.), and Discovery Biomarkers (P.T., S.M.), Pfizer Global Research & Development, Pfizer, Inc., Groton, Connecticut

Received for publication November 12, 2007
Accepted March 5, 2008.

	Abstract

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Among the L-type calcium channel blockers (CCBs), particularly dihydropyridines like nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], a common adverse effect is vasodilatory edema. Newer CCBs, such as the T- and L-type CCB, mibefradil [(1S,2S)-2-[2[[3-(2-benzimidazolylpropyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate], demonstrate antihypertensive efficacy similar to that of their predecessors but seem to have a reduced propensity to cause edema. Using a magnetic resonance imaging (MRI) T₂ mapping technique, we investigated the ability of mibefradil to reduce extracellular water accumulation caused by the L-type CCB, nifedipine, in the hindleg skeletal muscle of the spontaneously hypertensive rat. Mibefradil (10 mg/kg i.v.) and nifedipine (1 mg/kg i.v.) lowered mean arterial blood pressure by 97 ± 5 and 77 ± 4 mm Hg, respectively. MRI edema index (expressed as percentage increase of integral T₂ over predrug control) was significantly higher with nifedipine (2606 ± 86%; p < 0.05) than with mibefradil (981 ± 171%) measured 30 to 60 min after the start of drug infusion. The hindleg edema caused by nifedipine was dose dependently decreased by coadministration of mibefradil (0, 0.3, or 3 mg/kg). The hindleg edema formation was not due to albumin leakage into the interstitial space based on immunostaining. However, a 4.2-fold increase in the arterial L-/T-type CC mRNA expression ratio was observed compared with the venous L/T ratio as shown by quantitative reverse transcription polymerase chain reaction. These results demonstrate the novel utility of MRI to measure extravascular water after acute exposure to CCBs and indicate that T-type CCB activity may reduce L-type CCB-induced vasodilatory edema in the skeletal muscle vasculature, possibly by a differential effect on arteriole and venule dilatation.

Not all CCBs seem to have the same propensity for causing edema. For example, in the COHORT study, two lipophilic CCBs, lercanidipine and lacidipine, in elderly hypertensives caused edema in 9 and 4% of patients, respectively, compared with 19% for amlodipine (Leonetti et al., 2002). The mechanism for the reduced incidence of edema with more lipophilic CCBs like lercanidipine, lacidipine, and mibefradil still remains unclear.

Mibefradil (Brogden and Markham, 1997), which is structurally and pharmacologically different from traditional calcium antagonists, recently has been shown in clinical studies to reduce the incidence of leg edema to 5% compared with 26 and 17% for the dihydropyridines, amlodipine and nifedipine, respectively (Kobrin, 1997). Mibefradil represents a new generation of CCBs that blocks both the T- and L-type Ca²⁺ channels, whereas the dihydropyridines selectively block L-type Ca²⁺ channels. Previous work has shown that the L-type Ca²⁺ channel binding affinities for nifedipine and mibefradil, measured at the 1C subunit, had a K_i value of 8.2 and 156 nM, respectively (Huber et al., 2000). The T-type Ca²⁺ channel (i.e., the 1H subunit) binding affinities determined electrophysiologically for nifedipine and mibefradil had IC₅₀ values of >10 µM and 140 nM, respectively, with mibefradil having a 10 to 15-fold preference for the T-type versus the L-type Ca²⁺ channel (Martin et al., 2000). These reported Ca²⁺ channel affinity data illustrate the difference in binding capabilities between nifedipine and mibefradil and establish the proposed mechanism in the edema formation between nifedipine and mibefradil.

To explain the functional differences of T- and L-type Ca²⁺ channels in vascular tissue, previous studies have demonstrated the differential dilatory effects of CCBs on arteries and veins (Harris et al., 1980; Magnon et al., 1995; Ozawa et al., 2001). Feng et al. (2004) have recently shown that CCBs with combined T- and L-type activity such as pimozide and mibefradil equally dilate efferent and afferent arterioles in the renal circulation and provide protection against hypertensive glomerular injury, unlike L-type CCBs. These studies are consistent with the notion that CCBs with T-type activity have an attenuated ability to cause peripheral edema due to their equal vasodilatory effects on pre- and postcapillary vessels. The molecular mechanism involved may be dependent on the differential T- and L-type Ca²⁺ channels in arterial and venous tissue.

In this study, we examined whether MRI could be used to quantitatively detect CCB-induced peripheral edema in a well characterized model of hypertension, the spontaneously hypertensive rat (SHR), compared with the normotensive rat. After establishing that peripheral edema can be measured by this technique, we examined whether mibefradil could attenuate edema caused by nifedipine. The functional MRI results indicate that, at equally antihypertensive doses, mibefradil caused less vasodilatory edema than nifedipine, which is consistent with a mechanism that may equalize hydrostatic pressure across the capillary bed by decreasing venous resistance. This equalized capillary hydrostatic pressure may be due to either directly having equal T-type Ca²⁺ channel expression on the arterial and venous sides of the capillary or indirectly through a simple reduction in L-type Ca²⁺ channel expression on the arterial side.

	Materials and Methods

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Materials. Mibefradil and nifedipine were obtained from Sigma-Aldrich (St. Louis, MO). Intravenous solutions of both drugs were prepared using a cardiovascular-inert vehicle consisting of 5% N-methylpyrrolidone/45% polyethylene glycol 400/50% 50 mM lactic acid.

Animal Preparation. The animal handling and imaging procedures were approved by the Pfizer Institutional Animal Care and Use Committee according to the Institute of Laboratory Animal Resources (1996). Thirty-eight SHRs (360 ± 65 g) and six Sprague-Dawley (SD) normotensive rats (450 ± 50 g) were anesthetized with isoflurane in O₂ [3% (v/v) induction in a chamber followed by 1–1.5% (v/v) maintenance via a nose mask]. Catheters (PE50) were surgically placed into the left carotid artery (for blood pressure measurement) and the right jugular vein (for drug delivery). The animal was then placed supine on a heated bed, and the hindpaws were secured to the bed. The rat was allowed to breathe spontaneously. Respiration and mean arterial blood pressure (MBP) were monitored throughout the experiment with a chest pneumatic transducer and a blood pressure transducer connected to an animal monitoring system. Temperature was measured with the rectal fluoroptic thermometer and maintained at 36 ± 1°C.

Magnetic Resonance Imaging Protocol. AT₂ mapping MRI technique was adapted for the present studies (Patten et al., 2003). In brief, MRI was performed using a 7-Tesla Bruker MRI system. A 72-mm volume coil (Bruker Biospin, Billerica, MA) was used for radio frequency (RF) excitation and reception. Preliminary experiments were performed on phantoms to optimize the sequence for T₂ measurement. A vial with water titrated with gadopentetate dimeglumine (Magnevist; Berlex Pharmaceuticals, Wayne, NJ) to a known T₂ (served as a quality control imaging phantom) was placed between the hindpaws of the rat.

A multislice multiecho spin echo sequence was used to obtain axial images of the upper hindleg region for the calculation of the T₂ maps. The imaging parameters were as follows: matrix size, 128 x 128; field of view, 5.5 x 5.0 cm; 16 slices; slice thickness, 2 mm; echo time, 7 ms; 12 echoes; 2500-ms repetition time; and two averages. The edge of the slice pack was carefully positioned at the center of the knee joint, which served as a fiducial mark for reproducible slice planning. Each T₂ map acquisition took approximately 11 min.

Experiment Protocol. To test for the edema-generating mechanism of the L-type Ca²⁺ channel, nifedipine, both hypertensive SHRs as well as the normotensive SD rats were studied for MRI-measured hindleg edema, femoral artery/vein mRNA, and extracellular skeletal muscle albumin staining. In another set of experiments, the pharmacological effect of the mixed L- and T-type CCB, mibefradil, was assessed both alone and in combination with nifedipine to assess the influence of the T-type Ca²⁺ channel expression on peripheral edema formation in the SHR. MBP was allowed to stabilize for 30 min. The experiment was aborted if the predrug control mean MBP was greater than 130 mm Hg for the SD rat or less than 130 mm Hg for the SHR. Two T₂ map scans, 9 min apart, were acquired to establish the baseline condition. If the baseline was not achieved (i.e., there were statistical differences between average T₂ values in these two scans), the animal was excluded from experiment in the postprocessing stage. The rat was then dosed i.v. with nifedipine (1, 0.1, or 0.01 mg/kg; n = 3 rats/dose), mibefradil (10, 1, or 0.1 mg/kg; n = 3 rats /dose), or vehicle (1 ml/kg bolus + 1 ml/kg/60 min; n = 3 rats). The compound was delivered as an i.v. loading dose over 2 min, immediately followed by a maintenance infusion for over 60 min using a syringe infusion pump. Two T₂ map scans were acquired, one at 30 and 50 min after the start of the infusion, respectively. These scans from the drug infusion were averaged together for comparison to the predrug control scans. The blood pressure lowering was determined as the difference between baseline and a point with a minimal blood pressure, which invariably occurred right at the end of loading dose delivery.

The protocol for the combination of mibefradil with nifedipine involved the same MRI scanning paradigm as described above. The dose selection criteria for the combination of nifedipine and mibefradil were determined in separate experiments (data not shown) that demonstrated low, medium, and maximal blood pressure lowering.

MRI Data Analysis. Data were zero-padded to matrix size of 256 x 256 and analyzed using the AFNI software package (National Institutes of Health, Bethesda, MD). T₂ maps were calculated by monoexponential fitting of multiecho images using the least square error fitting. Percent increase in T₂ during drug treatment was calculated pixel-by-pixel. To limit the number of false positives, all increases in T₂ of less than 10% were set to zero. Regions of interest (ROI) were drawn on the muscle tissue on seven contiguous slices on anatomical images 8 mm distal from the knee joint (tibial plateau), which served as a fiducial mark. The stack of these ROIs was treated as a single volume ROI. Regions with bone structures (tibia and fibula) were excluded from ROI. The number of nonzero voxels (N) and the average change in T₂ (T₂) over these voxels was calculated in a volume ROI. The integral increase in T₂ (I = N xT₂) was used as quantitative biomarker for the severity of edema (MRI edema index). This could also be described as the sum of T₂ of all voxels within volume ROI, which changed for more than 10%. The quality control phantom had an average T₂ value of 44 ms. The data set was considered good for further analysis if there were no voxels in the phantom showing change in T₂ more than 10% during a single experiment.

The AFNI software package was used for volume measurements of the hindleg skeletal muscle in selected data sets. For volume calculations, two-dimensional ROIs were manually drawn on the anatomical images to cover the same regions (i.e., the same number of slices) that were analyzed for T₂ maps. The best judgment of human operator was used to discriminate between muscle and nonmuscle tissue. Volume was calculated as the sum of the ROI areas. The percentage increase in muscle volume was calculated from the predrug and postdrug scans.

Immunohistochemistry. In a separate set of animals for immunohistochemistry, formalin-fixed, paraffin-embedded sections were used for each SHR proximal hindleg skeletal muscle specimen to assess whether extracellular protein accumulation induced by the L-type CCB, nifedipine, caused hindleg edema. In brief, 1 mg/kg nifedipine or vehicle was delivered as an i.v. loading dose over 2 min, immediately followed by a maintenance infusion for 30 min using a syringe infusion pump to three SHRs. Animal euthanasia was immediately performed after the i.v. infusion of nifedipine or vehicle. For comparison, the same muscle masses in three normotensive Sprague-Dawley rats were used as normal skeletal muscle specimens. The tissue blocks were sectioned at 3-µm thick, placed on charged slides, and immunohistochemically stained on a Ventana Discovery Autostainer. Unless specified, all reagents are from Ventana Medical Systems (Tucson, AZ). Slides were deparaffinized, blocked for 30 min in Dako serum-free blocking solution (X0909; Dako North America, Inc., Carpinteria, CA) incubated in rabbit anti-human albumin antibody (A0001, 1:4000; Dako North America, Inc.) for 1 h at 37°C, blocked with avidin and biotin blocking reagents for 18 min each, then incubated in a biotinylated, anti-rabbit link (H+L) (BA-1000, 1:1000; Vector Laboratories, Burlingame, CA) for 30 min at 37°C. Slides were developed with a diaminobenzidine kit and counterstained with hematoxylin and bluing reagent for 4 min each, removed from the Autostainer, dehydrated, and coverslipped with Permount (Fisher Scientific Co., Pittsburgh, PA).

Real-Time Polymerase Chain Reactions. TaqMan real-time RT-PCR was performed to evaluate the mRNA expression levels in the major vessels that supply the hindleg skeletal muscle, namely the femoral artery and vein. These large vessels were used so that enough mRNA for expression work could be obtained for vascular tissue that perfuses a majority of skeletal muscle. The validation that the femoral vessels can serve as tissue surrogates for the pre- and postcapillary vessels on a molecular level has not directly been demonstrated, but pharmacologically, the conduit femoral arteries have been shown to function similarly to the small femoral arteries (200–300 µm) (Li and Schiffrin, 1996). One microgram of each total RNA sample was used to generate cDNA using the High Capacity cDNA Archive Kit following the manufacturer's protocol (Applied Biosystems, Foster City, CA). PCR assays were performed to measure the expression levels of two differentially expressed genes using rat sequence-based TaqMan Gene Expression assays on an ABI Prism 7700 Sequence Detection System (Applied Biosystems). The forward and reverse primers for the L-type Ca²⁺ channel subtype 1C (CACNA1C; amplicon = 236 bp) were 5'-CAAGAGTTGGTGGAGAAGCC-3' and 5'-TGAAGCTCAGAGAGTGGTCG-3', respectively. The forward and reverse probes for the T-type Ca²⁺ channel subtype 1H (CACNA1H; amplicon = 177 bp) were 5'-TCGAGGAGGACTTCCACAAG-3' and 5'-TGCATCCAGGAATGGTGAG-3', respectively. The fluorescence TaqMan probe contained a 6-carboxy-fluorescein phosphoramidite (FAM dye) label at the 5' end of the gene and a minor groove binder and nonfluorescent quencher at the 3' end designed to hybridize across exon junctions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA expression levels were used for normalization. For each target gene, cDNA corresponding to 20 ng of RNA was used per 20-µl reaction. Each reaction was performed in duplicate on a 384-well plate using the standard conditions determined by the manufacturer. The level of expression was calculated based upon the PCR cycle number (Ct) at which the exponential growth in fluorescence from the probe passes a certain threshold value. Relative expression was determined by the difference in the Ct values for the target genes after normalization to RNA input level, using GAPDH ribosomal RNA Ct values (Ct = Ct of the target gene minus Ct of the internal control GAPDH ribosomal RNA). Relative quantification between different samples (e.g., arterial versus venous) was determined according to the 2 - Ct method (Ct = Ct SHR sample - Ct normotensive sample) (Livak and Schmittgen, 2001).

Statistical Analysis. Data are expressed as mean ± S.E.M. Comparison between vehicle- and drug-treated groups was analyzed by the Student's t test method. Values of p < 0.05 were considered statistically significant. For both the mRNA expression and mibefradil/nifedipine combination studies, differences were evaluated using a one-way analysis of variance with the Hsu's mean comparison with the best (Hsu, 1996).

	Results

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Effects of Nifedipine and Mibefradil on Edema as Measured by MRI. To ascertain whether MR imaging is capable and sensitive to measure static, interstitial water as a marker for peripheral edema, the L-type CCB, nifedipine (total dose after 60 min equals 1 mg/kg), which is known to elicit peripheral edema, was assessed by T₂ mapping in the rat hindleg skeletal muscle. As shown in Fig. 1, representative T₂ maps after 60 min of nifedipine constant i.v. infusion show that nifedipine increases the T₂ values measured in a 7-Tesla MRI system compared with the vehicle. The T₂ change became measurable after only 30 min into the nifedipine infusion and continued to increase slightly during the following 20 min, whereas MBP dropped immediately after bolus dose delivery and gradually recovered during infusion of the rest of the compound (Fig. 2).

View larger version (63K): [in this window] [in a new window]   Fig. 1. Representative MRI T2 maps for vehicle and the L-type CCB, nifedipine, in SHR hindleg muscles. A, T2-weighted images before and 50 to 60 min after the i.v. administration of 1 mg/kg nifedipine. The T2 is calculated as the percentage change in T2 relaxation time measured before and during compound infusion based on the image signal intensity decay over 12 echoes with an MRI echo spacing equal to 7 ms. The third panel from left is a T2 map image showing the extracellular water accumulation levels with the green-blue areas the highest water content (100%) and the orange-red the lowest level of water accumulation (0%). B, similar T2-weighted images and T2 maps as in A but with the vehicle control. Notice that no T2 changes occurred with the vehicle. A phantom was used in MR imaging to ensure stability of T2 measurements during each single experiment.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Time course of MBP (solid line, left y-axis) and ROI averaged T2 (dashed line, right y-axis) in SHRs after infusion of nifedipine (1 mg/kg, black circles) and mibefradil (10 mg/kg, open circles). The beginning of loading dose delivery is marked as zero time point. Data are means ± S.E.M.

To confirm that the observed increase in the integral T₂ reflects edema, we measured the volume of muscle tissue on the same MRI scans we used for T₂ calculations in both the vehicle- and nifedipine-treated groups, which represent two extremes, baseline and positive control. The muscle volume did not change in the vehicle-treated group (1.5 ± 1.6%, p = 0.389 compared with zero) and increased in nifedipine-treated group (7.4 ± 1.3%, p = 0.031 compared with zero, p = 0.041 compared with the vehicle group). The correlation coefficient between integral T₂ and the volume changes were statistically significant (r = 0.741, p = 0.033; Fig. 3). Based on this strong correlation, we used the integral T₂ increase values as a surrogate for peripheral edema.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Correlation between integral T2 change and muscle volume change after infusion of vehicle (open circles) and nifedipine (1 mg/kg, black circles). Correlation coefficient (r = 0.741, p = 0.033) was computed with the Pearson Product Moment statistic.

Edema Assessment in SHR Skeletal Muscle at Equal Blood Pressure-Lowering Doses for Nifedipine or Mibefradil. The average predrug mean MBP across all the SHRs was 141 ± 3 mm Hg. Nifedipine and mibefradil each decreased mean MBP in a dose-dependent manner with nifedipine being approximately 10 times more potent than mibefradil (Fig. 4A) The mean MBP decrease caused by 1 mg/kg nifedipine (-77 ± 4 mm Hg; p < 0.05) was not statistically different compared with 10 mg/kg mibefradil (-97 ± 5 mm Hg).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Effects of nifedipine and mibefradil on SHR blood pressure and increase in percentage integral T2 (N x average T2) as an MRI index of peripheral edema. A, MBP-lowering effects of nifedipine (0.01, 0.1, and 1 mg/kg) and mibefradil (0.1, 1, and 10 mg/kg) in the anesthetized SHR. Nifedipine has 10-fold greater potency in BP lowering than mibefradil. B, dose-response to nifedipine (0.01, 0.1, and 1 mg/kg) and mibefradil (0.1, 1, and 10 mg/kg) on SHR hindleg edema formation. Nifedipine has a 2.5-fold greater propensity to form edema than mibefradil. The data in both A and B are means ± S.E.M. *, significant difference from vehicle group, p < 0.05.

Compared with the vehicle group, the edema formation during the 30 to 60-min i.v. infusion of nifedipine and mibefradil showed a dose-dependent increase, but the edema caused by mibefradil was significantly (p < 0.05) less than the edema caused by equal or less effective antihypertensive doses of nifedipine (Fig. 3B). At similar mean BP lowering, nifedipine at 1 mg/kg increased MRI edema index in the SHR hindleg muscle to 2606 ± 86%, whereas the high dose of mibefradil (10 mg/kg) increased the MRI edema index to 981 ± 171% (p < 0.05 between treatments; Fig. 4B). When compared with the vehicle, the 0.1 mg/kg dose of nifedipine and 1 mg/kg mibefradil significantly increased MRI edema index (1301 ± 419 and 554 ± 129%; p < 0.05, respectively), whereas the low doses of either drug did not increase MRI edema index in the SHR hindleg muscle.

In addition, the capacity of nifedipine to induce peripheral edema was compared between the SHR and a normotensive rat model where antihypertensives have been shown to exert attenuated activity. As shown in Table 1, the BP-lowering effect of 1 mg/kg nifedipine was significantly less in the normotensive SD rat when compared with the same dose in the SHR. The MRI edema index was significantly less in the normotensive rat compared with the SHR.

Albumin Expression in SHR Hindleg Muscle Specimens after Nifedipine Infusion. Peripheral edema can be caused by either an increase in capillary hydrostatic pressure forcing water into the extracellular space or by increased extracellular osmotic pressure due to increased protein leakage through the capillary wall. To rule out that protein extravasation into the extracellular space of the SHR hindleg muscle was possibly the mechanism for the rat hindleg edema, immunostaining for albumin in the skeletal muscle formalin-fixed sections was used. As shown in Fig. 5, C and D, no albumin was present in the extracellular space of the muscle after a 30-min nifedipine (1 mg/kg) or vehicle i.v. infusion in the SHR. The 30-min infusion was used because MRI studies have shown that there was edema developed already at this time point.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Immunostaining for albumin shows no protein in the rat hindleg muscle interstitial space. Albumin expression was determined by immunohistochemistry. Representative images of immunostaining for both formalin-fixed, paraffin-embedded normotensive Sprague-Dawley (A and B) and SHR (C and D) hindleg skeletal muscle. Arrows, specific albumin staining (dark-brown areas) inside blood vessels as positive staining control for antibody. Vehicle control tissues for both animals (A and C) as well as the edema-inducing L-type CCB, nifedipine tissues (B and D) show no albumin staining in the muscle. The albumin-specific antibody was visualized by diaminobenzidine chromogen; magnification, 20x.

1H (a T-Type Ca²⁺ Channel Subunit) and 1C (an L-Type Ca²⁺ Channel Subunit) mRNA Expression in the Femoral Artery and Femoral Vein. The mRNA expression levels of the T-type Ca²⁺ channel subunit, 1H, and the L-type Ca²⁺ channel subunit, 1C, from arterial and venous tissues were analyzed by the TaqMan quantitative RT-PCR technique. In the SHR femoral artery, the -fold change (from GAPDH housekeeping gene) in 1C (Fig. 6A) was 5.3 ± 2.2, whereas the femoral vein 1C (Fig. 6B) -fold change was 1.4 ± 0.4. The -fold change in the T-type Ca²⁺ channel mRNA expression was 1.1 ± 0.1 in the SHR femoral artery versus 1.2 ± 0.1 in the SHR femoral vein. The arterial L-/T-type expression ratio is 4.2-fold compared with the venous expression ratio.

View larger version (17K): [in this window] [in a new window]   Fig. 6. 1H (a T-type Ca2+ channel subunit) and 1C (a L-type Ca2+ channel subunit) mRNA expression in the femoral artery and femoral vein of the rat. For quantitative RT-PCR data, the relative levels (-fold change) of the 1C (A) and 1H (B) mRNA expressions were normalized to the housekeeping gene coding for GAPDH ribosomal RNA. The data are means ± S.E.M. with significance determined at the p < 0.05 level using a one-way analysis of variance with mean comparison by Hsu's MCB. *, significance for 1C gene expression compared with normotensive SD rat femoral artery, SHR femoral vein, and normotensive SD rat femoral vein.

Effects of Mibefradil in Combination with Nifedipine-Induced Edema in SHR Skeletal Muscle. Because L-type Ca²⁺ channel expression is greater in the arterial tissue (consistent with a hydrostatic edema mechanism), and T-type expression is more prevalent in the veins, we thought that adding a T-type CCB might attenuate L-type-induced edema. To determine whether the mixed T- and L-type CCB, mibefradil, could attenuate the edema induced by the L-type CCB nifedipine, a combination i.v. infusion of nifedipine at 1 mg/kg plus varying doses of mibefradil (0, 0.3, or 3 mg/kg) was assessed for edema formation in the SHR hindleg muscle. Representative MRI T₂ maps for the nifedipine and mibefradil combinations are shown in Fig. 7. Figure 7A is the 1 mg/kg nifedipine dose alone and shows the greatest increase in integral T₂ (i.e., edema) compared with either the nifedipine plus 0.3 mg/kg mibefradil (Fig. 7B) or nifedipine plus 3 mg/kg mibefradil (Fig. 7C) combinations. Upon quantification of the increase in integral T₂, a dose-dependent attenuation of the level of edema measured by MRI in the SHR hindleg muscle was observed as shown in Fig. 7D. Nifedipine at 1 mg/kg plus vehicle elicited an edema response of 2606 ± 86%, whereas nifedipine plus 0.3 mg/kg mibefradil and nifedipine plus 3 mg/kg mibefradil significantly reduced edema index (1725 ± 306 and 1139 ± 279%, respectively). The maximal BP lowering of nifedipine alone, nifedipine plus 0.3 mg/kg mibefradil, and nifedipine plus 3 mg/kg mibefradil was -77 ± 4, -75 ± 3, and -100 ± 7, respectively. Only the nifedipine plus 3 mg/kg mibefradil combination produced MBP lowering that was significantly different from the other treatment groups.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Mibefradil attenuates edema formation induced by nifedipine. T2 maps were determined as increase of T2 from the baseline to 30 to 60 min into the i.v. coinfusion of 1 mg/kg nifedipine plus vehicle (A), 1 mg/kg nifedipine plus 0.3 mg/kg mibefradil (B), and 1 mg/kg nifedipine plus 3 mg/kg mibefradil (C). The highest level of T2 change is depicted in the orange-red colors (100%), whereas the lowest T2 change is shown in green-blue colors (0%). The highest integral T2 increase was observed with the nifedipine/vehicle group and the lowest integral T2 change with the nifedipine plus mibefradil (3 mg/kg). D, dose-response to mibefradil (0, 0.3 and 3 mg/kg) in attenuating SHR hindleg edema formation induced by 1 mg/kg nifedipine. Mibefradil at 3 mg/kg was able to reduce the edema formed by nifedipine by approximately 2.5 times compared with the nifedipine/vehicle combination. The data are mean ± S.E.M.; 1N + 0M = 1 mg/kg nifedipine plus vehicle, 1N + 0.3M = 1 mg/kg nifedipine plus 0.3 mg/kg mibefradil, 1N + 3M = 1 mg/kg nifedipine plus 3 mg/kg mibefradil. *, significance of 1N + 0.3M and 1N + 3M versus 1N + 0M, p < 0.05.

	Discussion

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Peripheral edema is clinically characterized by a diffuse swelling in the lower extremities and is the most frequent adverse effect reported by patients receiving CCBs. The reported incidence of swollen ankles has been the major measurement for edema due to CCB treatment. However, determining edema comes late in the drug discovery process and renders a large risk to development of safer CCBs. Objective methods for measuring edema involve the evaluation of foot-ankle volume by water displacement. The changes in foot volume are greater with the less lipophilic CCBs nifedipine and amlodipine than with more lipophilic CCBs manidipine or lercanidipine (van Hamersvelt et al., 1996; Lund-Johansen et al., 2003). However, only large changes in foot-ankle volume can be detected. A more sensitive measurement of edema earlier in the preclinical and clinical evaluation would enable the discovery and development of safer antihypertensive agents.

The ability to assess edema by MRI currently exists in the hospital setting because most MRI practices are able to perform the T₂ mapping for detecting many pathological conditions, including muscle edema (May et al., 2000; Patten et al., 2003). Ababneh et al. (2005) have shown that the T₂ proton relaxation can be fit with two exponential decay components, one short and another long. These components assumably correspond to intracellular (short decay component) and extracellular water (long component). It was found that edema had a marked dependence on an increase in the T₂ long decay component, whereas exercise moderately changed the T₂ short decay component (Ploutz-Snyder et al., 1997). We used a single-exponential model for T₂ relaxation fit for simplicity. During method development (data not shown), we found that the number of refocusing (180°) RF pulses should be limited to <16 due to RF power deposition and subsequent heating of the subject, which may affect T₂ measurement significantly. We have observed a consistent change in T₂ values measured with a monoexponential fit after the induction of edema (from 31.2 ± 0.7 to 44.8 ± 1.0 s after 60-min infusion of 1 mg/kg nifedipine). To increase the dynamic range and sensitivity of the measurement, we combined both the intensity of the change in T₂ (T₂) and the affected area (N, number of pixels with T₂ > 10% over baseline) to derive integral T₂ parameter or MRI edema index. Although T₂ changes were not quite reaching the plateau 50 min after drug infusion (see Fig. 2), we chose to use predetermined time points to quantify the MRI edema index rather than wait for T₂ stabilization. To increase the throughput and decrease the effect of anesthesia on edema, we decided to limit scanning to 60 min after drug infusions. To validate, at least partially, the use of T₂ mapping for detecting edema, we have measured the changes of muscle volume in response to the CCB treatment. These changes were very subtle and detectable only for two extreme cases: the vehicle- and nifedipine-treated groups. There was a significant correlation between changes in muscle volume and integral T₂ values, which confirms the specificity of T₂ mapping method to measure edema. However, the sensitivity of the integral T₂ is greater by several orders of magnitude. It should be noted that the change in volume of the muscle is the potential source of error in estimation of integral T₂ change because it is based on pixel-by-pixel comparisons. The pixels in the edematous images are slightly shifted compared with the baseline. However, the extent of the linear shift is very small because the volume of the muscle changed for only 7.4% in the extreme case. It should have not changed pixel-by-pixel comparisons significantly.

The next question was whether integral T₂ increase is sensitive enough to differentiate between the edema-causing dihydropyridine CCBs (nifedipine) and the mixed T- and L-type CCB, mibefradil. This study describes the novel utility of MRI in differentiating between compounds by measuring their ability to produce edema. A mechanistic limitation inherent in this study stems from the lack of a selective T-type Ca²⁺ CCB. Even though mibefradil has T-type Ca²⁺ channel-blocking activity, it also has L-type CCB activity that can confound data interpretations. However, our results implicate that the lessening of the L-type CCB activity and/or increasing the T-type CCB activity can reduce peripheral edema.

Edema formation is increased by nifedipine in the SHR hindleg muscle in a short period of time (i.e., <1 h). A possible mechanism for the increase in extracellular water in the skeletal muscle involves an increased capillary hydrostatic pressure rather than extravasation of serum protein because immunostaining for albumin in muscle was negative. The ability of mibefradil to attenuate edema induced by the nifedipine may indicate a differentiating mechanism in the vasodilatory effects of the two CCBs on precapillary arterioles and the postcapillary venules. This study suggests that acute water accumulation in skeletal muscle is a hemodynamic mechanism versus any vessel wall restructuring, i.e., albumin leakage. The mRNA results for 1H and 1C differential expression demonstrating a 4.2-fold increase in the arterial L-/T-type mRNA expression ratio versus veins are consistent with the hypothesis that peripheral edema caused by CCBs results from selective arterial dilation and increased capillary hydrostatic pressure. The differential inhibitory effects between arteries and veins of various CCBs have been demonstrated where the L-type CCBs dilated arterioles more than venules (Harris et al., 1980; Magnon et al., 1995; Ozawa et al., 2001). Even though the differential T- and L-type Ca²⁺ channel expression between the femoral artery and vein in this study is on major vessels, the results confirm the published differences of particularly the L-type Ca²⁺ channel involvement in dilating the precapillary side to a greater extent than the postcapillary vessels (Li and Schiffrin, 1996). To support the hypothesis that the greater the L-type Ca²⁺ channel present on the arterial side of the capillary the more edema that could result was tested in normotensive SD rats. These studies confirmed that less edema developed in the normotensive rat compared with the SHR at the same dose of nifedipine. These data plus the differential L-type Ca²⁺ channel mRNA expression provide strong support of the degree of differential L-type Ca²⁺ channel between the arterial and venous sides that can drive the level of edema. This study has shown that extracellular protein accumulation is not driving the mechanism of CCB-induced peripheral edema and that L-type differential expression between the arterial and venous sides may actually play a large role in driving the peripheral edema formation due to L-type CCBs.

General anesthesia may have confounding effects on animals' BP and response to hypertensive therapy. However, it was shown that isoflurane had the least effect on cardiovascular parameters, especially if the minimal levels of anesthesia were used (Dardai and Heavner, 1987). These levels are what we used in this article. Because cardiovascular parameters drive the edema response the minimal effects should be seen on edema formation using isoflurane.

In summary, this study reveals for the first time that the MRI T₂ mapping provides a highly sensitive biomarker of edema, which could be used for drug research. Due to relative simplicity, this method has the potential for clinical translatability. At equal BP-lowering doses, compounds with both T- and L-type Ca²⁺ channel-blocking activity have significantly less increase in integral T₂ or edema formation than compounds with selective L-type Ca²⁺ channel-blocking activity. In addition, mibefradil can dose dependently attenuate the peripheral edema induced by nifedipine. This study may indicate that mixed T- and L-type CCBs may equalize the hydrostatic pressure across the capillary bed by equally dilating arterioles and venules, reducing vasodilatory edema. These findings suggest that the design of novel CCBs should incorporate a mix of T- and L-type Ca²⁺ channel activity to reduce the risk of edema. Thus, the development of a mixed T- and L-type CCB could replace the edema-laden L-type CCBs as antihypertensives.

	Acknowledgements

 
We thank David G. Taylor and Robert Leadley for scientific discussions and editorial assistance in preparing this manuscript.

	Footnotes

 
We declare no other sources of funding beyond that of Pfizer, Inc. We declare no conflicts of interest.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.133892.

ABBREVIATIONS: CCB, calcium channel blocker; nifedipine, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester; mibefradil, (1S,2S)-2-[2[[3-(2-benzimidazolyl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate; MRI, magnetic resonance imaging; SHR, spontaneously hypertensive rat; SD, Sprague-Dawley; MBP, mean arterial blood pressure; RF, radiofrequency; ROI, region(s) of interest; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ct, threshold cycle; BP, blood pressure.

Address correspondence to: Dr. Serguei Liachenko, Bioimaging Center of Emphasis, Pfizer Global Research and Development, Pfizer, Inc., Eastern Point Road 118W-339, Groton, CT 06340. E-mail: serguei.liachenko{at}pfizer.com

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