Figures
Abstract
5-[(4-carboxybutanoyl)amino]-2-hydroxybenzoic acid (C2) is a novel synthetic derivative of 5-aminosalicylic acid (5-ASA), which is currently being evaluated ex vivo as an anti-inflammatory agent and has shown satisfactory results. This study aimed to obtain the pharmacokinetic profiles, tissue distribution and plasma protein binding of C2 in Wistar Rats. Additionally, an HPLC method was developed and validated to quantify C2 in rat plasma. The pharmacokinetic profiles of intragastric, intravenous and intraperitoneal administration routes at singles doses of 100, 50, and 100 mg/kg, respectively, were studied in Wistar rats. The elimination half-life of intravenously administered C2 was approximately 33 min. The maximum plasma level of C2 was reached approximately 24 min after intragastric administration, with a Cmax value of 2.5 g/mL and an AUCtot value of 157 μg min-1/mL; the oral bioavailability was approximately 13%. Following a single intragastric or oral dose (100 mg/kg), C2 was distributed and detected in all examined tissues (including the brain and colon). The results showed that C2 accumulates over time. The plasma protein binding results indicated that the unbound fraction of C2 at concentrations of 1 to 20 μg/mL ranged from 89.8% to 92.5%, meaning that this fraction of C2 is available to cross tissues. Finally, the blood-plasma partitioning (BP ratio) of C2 in rat plasma was 0.71 and 0.6 at concentrations of 5 and 10 μg/mL, respectively, which indicates that C2 is free in the plasmatic phase and not inside blood cells. The results of this study suggest that a fraction of the administered C2 dose is absorbed in the stomach, and the fraction that is not absorbed reaches the small intestine and colon. This distribution constitutes the main advantage of C2 compared with 5-ASA for the treatment of ulcerative colitis (UC) and Crohn's disease (CD).
Citation: Romero-Castro A, Gutiérrez-Sánchez M, Correa-Basurto J, Rosales Hernández MC, Padilla Martínez II, Mendieta-Wejebe JE (2016) Pharmacokinetics in Wistar Rats of 5-[(4-Carboxybutanoyl)Amino]-2-Hydroxybenzoic Acid: A Novel Synthetic Derivative of 5-Aminosalicylic Acid (5-ASA) with Possible Anti-Inflammatory Activity. PLoS ONE 11(7): e0159889. https://doi.org/10.1371/journal.pone.0159889
Editor: Mária A. Deli, Hungarian Academy of Sciences, HUNGARY
Received: January 31, 2016; Accepted: July 8, 2016; Published: July 25, 2016
Copyright: © 2016 Romero-Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The authors thank the Consejo fNacional de Ciencia y Tecnología (CONACYT: I010/0532/2014; 132353), Instituto de Ciencia y Tecnología del Distrito Federal (ICyTDF/279/2011), Comisión de Operación y Fomento de Actividades Académicas/Secretaría de Investigación y Posgrado-IPN (COFAA-SIP/IPN: 20161383; 20160204; 20161374), and The Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED: 214RT0482) for their financial support. ARC and MGS thank CONACYT for their postdoctoral (290917-IPN) and M. Sc. scholarship (130245), respectively.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: 4-ABAH, 4-aminobenzoic acid; AD, Crohn's disease; 5-ASA, aminosalicylic acid; AUC0-t, area under the plasma concentration-time curve from time zero to the last measurable concentration; AUCtot, the area under the plasma concentration-time curve from time zero to infinity; BP ratio, blood-plasma partitioning; C2, 5-[(4-carboxybutanoyl)amino]-2-hydroxybenzoic acid; CL, clearance; Cmax, maximum plasma concentration; HOCl, hypochlorous acid; MRT, mean residence time; MPO, mieloperoxidase; HPLC, high performance liquid chromatography; RP-HPLC, reverse-phase high resolution liquid chromatography; SP, sulfapyridine; t1/2, elimination half-life; Tmax, time to reach Cmax; UC, ulcerative colitis; Vd, apparent volume of distribution
Introduction
Inflammatory bowel disease (IBD) is the medical term used to describe chronic inflammatory diseases of the gastrointestinal tract (GI) that are characterized by a wide range of signs and symptoms, such as diarrhea, abscesses, fistulas, abdominal pain and stenosis. These symptoms significantly affect the quality of life of affected patients [1, 2]. Although the symptoms of Crohn’s disease (CD) and ulcerative colitis (UC) are quite similar, they affect different areas in the GI are different. Specifically, CD most commonly affects the end of the small bowel (the ileum) and the beginning of the colon, but it may affect any part of the GI tract, from the mouth to the anus. Conversely, UC inflammation typically arises in the distal colon and extends in a proximal direction [3–6]. Both CD and UC are characterized by periods of active intestinal inflammation that may require hospitalization [1, 2, 5, 7, 8].
The main goal of pharmacological IBD treatment is the reduction of inflammatory process during relapses [9–13]. 5-aminosalicylates are non-steroidal anti-inflammatory drugs (NSAIDS) characterized by their analgesic, antipyretic, and anti-inflammatory effects. These drugs are used in conventional therapies to decrease the exacerbated immune responses in CD or UC in patients. Sulfasalazine, mesalazine, olsalazine, and balsalazide are 5-aminosalicylates that are used to treat UC, but their use in patients with CD remains controversial [13–17]. These compounds act locally on the colonic mucosa, but unfortunately, they are associated with side effects, such as diarrhea, nausea, vomiting, headache, abdominal pain, fatigue, weaknesses, hepatic abnormalities, arthralgia and myalgia. Specifically, the side effects of mesalazine (5-aminosalicylic acid) have been related to the sulfapyridine component [18, 19].
Uncoated 5-aminosalicylic (5-ASA) is absorbed in the small intestine when orally administered. Therefore, it cannot reach the colon mucosa in its unchanged form [20–24], and 5-ASA is consequently currently administered in oral formulations of delayed or controlled release and as a prodrug (sulfasalazine, olsalazine, balsalazide) [25–31].
Several research groups have designed new anti-inflammatory molecules that inhibit myeloperoxidase (MPO) [32]. Our group designed [33] and synthesized 5-[(4-carboxybutanoyl)amino]-2-hydroxybenzoic acid (C2) (Fig 1), which has shown satisfactory results in pharmacologic and toxicological studies [34–36]. Specifically, C2 inhibited the catalytic activity of MPO in a model of inflammation in the mouse ear [34] and was nontoxic in CD1 mice and Wistar rats (LD50 > 2000 mg/kg) [37]. In this work, the pharmacokinetic profiles (intragastric, intravenous and intraperitoneal) and distribution of C2 in rats were studied to assess the ability of C2 to reach the rat colon, which is a necessary condition for its local pharmacologic affect. To quantify C2 in the plasma, organs and tissues, a bio-analytical method was developed and validated using High Performance Liquid Chromatography (RP-HPLC).
Material and Methods
Chemicals and Standards
Acetonitrile, methanol and water (HPLC grade) were purchased from Tecsiquim (Mexico). Acetic acid (analytical grade), polysorbate 80 (tween) and propylene glycol were purchased from Sigma–Aldrich (St. Louis, MO, USA). Heparin (1000 UI/mL) and 0.9% sodium chloride were purchased from PISA (Mexico). The anesthetics ketamine (CLORKETAM®) and xylazine PROCIN®) were intended for veterinary use and acquired from Vétokinol (Lure Cedex, France) and PISA Agropecuaria, S.A. de C.V. (Hidalgo, Mexico), respectively.
A batch of C2 was synthesized at the Laboratorio de Investigación en Química Orgánica y Supramolecular de la Unidad Profesional Interdisciplinaria de Biotecnología; the molecular structure of C2 was validated using infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy and 13C and mass spectrometry; these analyses were conducted by the Centro de Nanociencias y Micro y Nanotecnología-IPN. The purity of C2 was assessed by HPLC. This batch of C2 was used as the reference standard due to its high purity (98.7%).
C2 Formulation for Administration
The intravenous formulation was a translucent white solution prepared by dissolving 100 mg of C2 in 10 mL of a mixture consisting of propylene glycol-polysorbate 80-sodium chloride (0.9%) at a ratio of 20:5:75, (v/v/v), respectively. The solution was vortexed for 3 min and sterilized by filtration before being filtered with a 0.22 μm nylon syringe filter; the final concentration was approximately 10 mg/mL.
Ethics Statement
The animal procedures were conducted in accordance with the Mexican Official Standard NOM-062-ZOO-1999, Technical Specifications for Production, Care and Use of Laboratory Animals [38]. The animal protocol was approved by the Research Committee for the Care and Use of Laboratory Animals (CICUAL) of the Escuela Superior de Medicina-IPN (Approval number: ESM.CICUAL-02/27-07-2015; S1 File).
In this study, the experiments were finished by the experimental endpoint. However, all animals were monitored for early indicators of a human endpoint [39]. After the collection of samples, the animals were sacrificed with an intraperitoneal dose of 72 mg/kg sodium pentobarbital.
Animals
Male Wistar rats were obtained from the Bioterio of the Escuela Superior de Medicina, Instituto Politécnico Nacional. For all experiments, the animals were placed in polycarbonate cages during the acclimation (one week) period and the experiments. The diet consisted of Rat Chow 5012 (Purina) and water ad libitum. For the pharmacokinetic studies, the animals were randomly divided into three groups (n = 6, body weight 300 ± 20 g). The rats were fasted for 8 h before dosing but allowed free access to water.
Rat Plasma Collection for Validation
Plasma was obtained from rats using a cardiac puncture protocol. Briefly, the animals were anaesthetized with sodium pentobarbital, and cardiac puncture was rapidly performed; the blood samples were then collected into heparinized tubes. The plasma was separated by centrifugation at 8,000 rpm and 4°C for 15 min and stored at– 80°C until use.
Instrumentation and Analytical Conditions
The levels of C2 were quantified using an Agilent 1260 Infinity Series liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary pump delivery system (G1311B), robotic autosampler (G1316A), column thermostat (G1316A), and multi-wavelength UV detector (G1315C); the results were analyzed using OpenLab CDS EZChrom. A Zorbax SB-C18 column (5 μm, 4.6 x 150 mm, Agilent Technologies, Palo Alto, CA, USA) was used for separation, and UV detection was carried out at 254 nm. The column temperature was maintained at 45°C, and the injection volume was 10 μL. The mobile phase consisted of a mixture of (A) 0.2% acetic acid in water (v/v), pH 3.0 and (B) acetonitrile at a ratio of 20% A and 80% B and flow rate of 0.6 mL/min. The total analysis time of each sample was 6.0 min using an isocratic elution, and equilibrating between each injection was not required.
Preparation of Calibration Standards and Quality Control (QC) Samples
Six stock solutions (1.0 mg/mL) were prepared by dissolving the appropriate weight of the compound in a mixture of methanol (50:50, v/v). These samples were stored at 2–4°C and were stable for one month. Calibration standards were prepared daily by diluting stock solutions. Briefly, 100 μL of blank plasma was spiked with an appropriate volume of stock solution, and the samples were then vortexed for 5 min. Next, appropriate volumes of acetonitrile-methanol (50:50) were added until each sample reached a final volume of 1 mL; these samples were centrifuged at 8,000 rpm for 10 min, and the supernatant was subsequently filtered using a nylon syringe filter with a 0.45 μm pore size. A volume of 10 μL was injected into the HPLC system. The standard solutions were prepared in triplicate for the eight calibration points, and the final concentrations obtained were 1, 2.5, 5, 10, 25, 50, 100, 250 and 500 μg/mL. Finally, calibration curves were constructed in triplicate by plotting known concentrations of the standard versus the detector response area.
A separate set of stock solutions was prepared for the QC samples for use in the method validation to assess accuracy and precision. All QC samples were prepared over three different concentration ranges in triplicate. The calibration and QC samples were prepared on different days. Furthermore, the QC samples were prepared independently using plasma from untreated rats.
Method Validation
The method was validated according to the FDA Industry Guidance for the Bioanalytical Method Validation and ICH Harmonised Tripartite Guideline for the Validation of Analytical Procedures [40, 41]. The following parameters were evaluated: linearity, intra- and inter-day accuracy and precision, recovery, the lower limit of quantification (LLOQ), the limit of detection (LOD), selectivity, and stability.
Linearity.
Linearity was determined as follows: A nine-point calibration curve was constructed by analyzing the standards in the concentration range mentioned above. Linearity was measured for the selected concentration range by fitting the data to a linear regression model and assessing the coefficient of determination (R2 ≥ 0. 980) [40, 41].
Intra- and Inter-Day Accuracy and Precision.
These parameters were determined by analyzing different QC samples (n = 5) at high, medium, and low concentrations of C2 (1, 25, and 100 μg/mL) on five consecutive days. The accuracy and precision are expressed in terms of the relative error (RE) and relative standard deviation (RSD), respectively. The intra- and inter-day precision should not exceed 15%, and the accuracy should be within ± 15% for the QC samples [40, 41].
Recovery.
The extraction recovery of C2 in rat plasma was assessed by comparing the mean peak areas of the processed QC samples with those of the corresponding the blank plasma matrix samples spiked with standard solutions, which were similarly prepared and had the same final concentration, except water replaced the blank plasma. The recovery of C2 was assessed at 1, 25, and 100 μg/mL [40, 41].
Lower Limit of Quantification (LLOQ) and Limit of Detection (LOD).
The LLOQ was determined from the last point of the calibration curve, and the average recovery value was ± 20% of the nominal value with a coefficient of variation (C.V.) ≤ 20%. The LOD was calculated according to the following equation: LOD = 3.3 σ/S, where σ represents the standard deviation of the intercepts of the regression lines, and S represents the mean of the slopes of the calibration curves of the analyte (ICH, 2005) [41].
Selectivity and Specificity.
Selectivity is the ability to accurately and specifically measure the analyte in the presence of components that may be expected to be present in the sample matrix. This parameter was investigated by analyzing blank plasma samples from six rats at the LLOQ concentration, which were then compared with the corresponding plasma samples spiked with C2 [40, 41].
In addition, we analyzed the potential interference of heparin, ketamine and xylazine, which were used in this pharmacokinetic study. Hence, the specificity was determined by assessing the peak identity and purity of C2. A peak purity angle that is less than the peak threshold angle is an indication of the spectral homogeneity or purity of C2.
Stability.
The stability of C2 in plasma was evaluated using the QC samples at two concentrations by triplicate (high = 100 μg/mL, medium = 25 μg/mL and low = 1 μg/mL); these samples were stored at 25°C for 12, 24 and 36 h (short-term stability) and subjected to three freeze-thaw cycles (–20°C to 25°C). The post-preparative storage was evaluated by analyzing the ready-to-inject samples that had been refrigerated 4°C for 12, 24 and 36 h. The concentration of C2 after each storage period was related to the initial concentration of freshly prepared and immediately processed samples [40, 41].
Pharmacokinetic Studies
Prior to the studies (on the day of C2 administration), the Wistar rats (n = 6) were cannulated. For intravenous (i.v.) treatment, a 24 G catheter was placed in the right lateral tail vein (for blood collection), and another catheter was placed in the left (for C2 administration) lateral tail vein of the rat as follows: the animal was placed inside a trap (special acrylic stocks for rats), the rat tail was exposed, and the distal region of the tail was disinfected with 70% ethanol; the tail veins were then dilated using a heating pad (approximately 45°C). An assistant held the tail of the rat while the left lateral vein was located, and a 24 G catheter was then firmly inserted into the posterior third of the caudal vein of the animal, where it was fixed with adhesive tape (for intragastric and intraperitoneal studies, only one catheter was placed on the right for blood collection).
For the intravenous study, a single dose of 50 mg/kg C2 was administered through the 24 G catheter when the rat was inside the trap. For the intragastric (i.g) pharmacokinetic study, a second group of rats (n = 6) received a single dose of 100 mg/kg using a 20 G gavage needle (animals were provided with a standard diet 4 h after dosing). For the intraperitoneal (i.p.) treatment, a third group of rats (n = 6) was injected with 75 mg/kg C2. In all pharmacokinetic studies, approximately 250 μL of blood was collected in heparinized tubes from each rat via the catheter in the right lateral tail vein prior to dosing and 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, 360, 540, 720 and 1440 min after dosing. These blood samples were supplemented with an equal volume of 0.9% sodium chloride solution. Plasma (100 μL) was harvested by centrifuging the blood samples at 8,000 rpm for 15 min, and the plasma was then immediately processed for analysis.
Sample Preparation.
The samples were prepared as follows: briefly, 450 μL of acetonitrile was added to 100 μL of rat plasma, and the mixture was vortexed for 5 min. Subsequently, 450 μL of methanol was added to the sample, which was then vortexed again and centrifuged to precipitate proteins. Subsequently, 10 μL of the supernatant was introduced into the HPLC system. The plasma concentrations of C2 were determined using an RP-HPLC with UV detection method that had previously been validated for pharmacokinetic studies and the complementary assays performed in this work.
Data Analysis.
The plasma concentration–time data obtained after the i.v., i.g., and i.p. administration of C2 were subjected to a non-compartmental analysis using statistical moment theory. The pharmacokinetic parameters of C2 were calculated using the Kinetica 5.0 software (Adept Scientific Ltd), including the maximum plasma concentration (Cmax), time to reach Cmax (Tmax), elimination half-life (t1/2), area under the plasma concentration-time curve from time zero to the last measurable concentration (AUC0-t), area under the plasma concentration-time curve from time zero to infinity (AUCtot), mean residence time (MRT), clearance (CL), and apparent volume of distribution (Vd).
Tissue Distribution Study.
Another group of fifteen male Wistar rats was orally administered a single 75 mg/kg dose of C2. The animals were sacrificed 10, 30, 90, 180 and 360 min after dosing (n = 3 per treatment time). The whole brain, heart, liver, spleen, lung, kidney, stomach, small intestine, colon, testicles and muscle were rapidly dissected, harvested and thoroughly rinsed in ice-cold saline to eliminate blood and other content. All tissues and organs were weighed on an analytical balance and immediately processed for analysis. Each tissue sample was homogenized with saline solution at a 1:3 (wt/v) ratio. The preparation process for analysis was the same as that described above for plasma [42].
Blood-Plasma Partitioning (BP Ratio).
Whole blood was spiked with different amounts of C2 to obtain final concentrations of 5 and 20 μg/mL, and these samples were then incubated at 37°C for 4 h (samples were prepared by triplicate). The plasma was separated from blood samples, and the concentration of C2 was determined based on a standard curve prepared with blank plasma using the RP-HPLC method. The blood/plasma concentration ratio (BP) was determined by dividing 5 and 20 μg/mL by the concentration found in plasma sample. The concentration of C2 in blood cells was assumed to be equal to its unbound concentration in the plasma [43].
Plasma Protein Binding Assay.
To assess the plasma protein binding of C2 using the ultrafiltration method, 600 μL samples of freshly obtained plasma from untreated rats (blank) were spiked with different amounts of C2 (10 mg/mL) to give final concentrations of 1, 5, 20 and 30 μg/mL. The resulting samples were incubated for 4 h at 37°C. After incubation, a 100 μL aliquot was removed to analyze the total concentration, and the remaining 500 μL was transferred to a 10 kD cut-off ultrafiltration device (Millipore Corporation, USA), which was centrifuged at 2,000 g for 2 h at 37°C. A 100 μL aliquot of centrifuged plasma was analyzed for the free drug concentration using the RP-HPLC method. The amount of the test compound in the ultrafiltrate was determined by interpolating the main areas of the samples on standard curves containing known amounts of C2. The percentage of protein binding was calculated using the following formula: Protein binding ratio (%) = [(1 –(drug ultrafiltrate)) / (drug total)] x 100. Additionally, the use of protein-free plasma instead of plasma indicated that C2 minimally bound to the ultrafiltration device [42, 43].
Results
Method Validation
The chromatographic conditions, especially the composition of the mobile phase, were optimized throughout several trials to achieve a good resolution and symmetric peak shapes for C2. Modifications, such as phosphoric acid and acetic acid alone or in combination at different concentrations, were tested. After testing several conditions, an isocratic elution of the mobile phase consisting of acetonitrile and 0.2% acetic acid (80:20) using a Zorbax SB C-18 column (150 mm × 4.6 mm, 5 μm) was found to yield a good peak shape and a suitable retention time of 10.2 min.
C2 was extracted to avoid plasma interference and achieve good and consistent recovery. Conventional methodologies for protein precipitation were tested; protein-precipitating agents, such as acids, methanol, chloroform, ethyl acetate, and dichloromethane alone or in combination at different proportions, were investigated to efficiently extract C2. We developed a two-step liquid-liquid extraction procedure from plasma using 450 μL of acetonitrile and 450 μL of methanol versus 100 μL of plasma, which excellently recovered C2 (above 90%): a clean chromatogram for a blank plasma sample and a high recovery of C2 from plasma were obtained.
Linearity.
All plasma calibration curves for C2 were linear over the concentration range of 1 to 500 μg/mL (Fig 1) with a correlation coefficient (R) greater than 0.99. A typical linear regression equation for the calibration curve was y = 59390x + 256011 (R2 = 0.9990), where y represents the area of the peak of C2 and x represents the concentration of C2 in the sample.
The values of the LLOQ and the LOD were found at 1 μg/mL for both parameters with a C.V. of 5.9%.
Intra- and Inter-Day Accuracy and Precision.
The intra- and inter-day accuracy and precision parameters at 1, 25, and 100 μg/mL of C2 are shown in Table 1. The intra-day precisions varied from 2.4% to 5.4, and the accuracies varied from 99.2% to 95.6%. The inter-day precisions varied from 0.7% to 4.2%, and accuracies varied from 86.5% to 96.2%. Thus, the intra-assay and inter-assay accuracy and precision were found to be acceptable and satisfactory for the C2 analysis, in support of further pharmacokinetic studies. The data demonstrated good accuracy and reproducibility, which indicated the applicability of the method to pharmacokinetic studies.
Recovery.
The absolute recoveries of C2 determined at three concentrations, 1, 25 and 100 μg/mL, were 96.0%, 105.7% and 108.9%, respectively. The absolute percent recovery values of C2 are summarized in Table 2.
Selectivity.
C2 was successfully extracted from spiked rat plasma and chromatographically resolved using the previously described sample preparation procedures and chromatographic conditions. Typical chromatograms obtained after the analysis of the C2 of blank and spiked rat plasma samples are shown in Fig 2A and 2B, whereas Fig 2D shows a chromatogram of an in vivo plasma sample from a rat that had been administered C2. The retention time of C2 was approximately 2.2 min. The chromatograms in the blank rat matrix showed that no endogenous compounds interfered with the retention times of C2, which indicates that the assay was selective. In addition, under the described chromatographic conditions, none of the tested drugs (ketamine, xylazine and heparin) that are frequently used in experimental protocols of pharmacokinetic studies were found to interfere with the retention time of the chromatographic peak of C2 Fig 2C.
(A) Biological matrix (plasma) chromatogram. (B) Chromatogram of 1 μg/mL C2 standard. (C) Chromatogram of biological matrix spiked with ketamine, xylazine and heparin (D) Chromatogram of an in vivo plasma sample from a rat that had been administered C2.
Stability.
The stability of C2 was investigated under a variety of conditions at four concentrations (1, 2.5, 25 and 100 μg/mL), which were assessed in triplicate, and the results are summarized in Table 3 (stability sample results should be within 15% of nominal concentrations). The data showed that C2 was stable in rat plasma stored at 4°C and 25°C for 36 h, and it was also stable before the three freeze–thaw cycles of the quality control samples.
Pharmacokinetic study
After the intravenous administration of 50 mg/kg of C2, the plasma concentration declined rapidly (Fig 3A). The MRT value for C2 was 57 ± 11 min, the t1/2 was approximately 33 ± 5 min, and the Vd was 2.0 ± 0.2 L/kg. After oral dosing (100 mg/kg), C2 was absorbed, and the maximum plasma levels reached a peak at 24 min (Fig 3B), with a Cmax value of 2.5 ± 0.3 μg/mL and a t1/2 of approximately 27 ± 11 min. The absolute oral bioavailability of C2 was only 13%. The values of all pharmacokinetic parameters for each route of administration are shown in Table 4. As shown in Fig 3, C2 was poorly absorbed at the tested dose (100 mg/kg), reaching maximum plasma levels within 2.5 min and 8.7 min of i.g. (Fig 3B) and i.p. (Fig 3C) administration, respectively. As a consequence, the Cmax and AUCtot were higher for the i.v. route than i.p. and i.g. routes at the lower dose (Table 4).
Values are presented the as the mean ± SD.
Subsequently, the software Kinetica 5.0 was used to establish the pharmacokinetic model of C2, which showed that the plasma concentration declined bi-exponentially. Specifically, the parameters α = 0.037 and β = 0.011 min-1 were determined, and the values K12 = 0.008 min-1 and K21 = 0.022 min-1 were calculated; the elimination half-life α was t1/2α = 18 min, and the elimination half-life β was t1/2β = 62 min
Tissue Distribution.
This study assessed two conditions, i.g. (Fig 4) and i.p. (Fig 5), at different time points (10, 30, 60, 120 and 360 min). Before the 2 h time point, C2 was widely distributed in all examined tissues for both administration routes (i.g., and i.p.), and the maximum concentration decreased throughout the 6 h of monitoring. The results show the presence of C2 in the brain and testicles, indicating that C2 effectively crossed the blood-brain barrier and hemato-testicular barrier. For routes of administration, C2 tended to accumulate in almost all tissues examined. After i.g. administration, a higher concentration of C2 was detected in the liver, and a similar concentration of C2 was detected in the stomach; moreover, i.g. administration resulted in lower C2 concentrations in the colon and small intestine than i.p. administration, suggesting that the stomach is the absorption site of C2.
(Mean ± SD, n = 3).
(Mean ± SD, n = 3).
Protein binding and Blood-Plasma partitioning (BP ratio).
The protein-binding data of C2 are summarized in Table 5. The unbound fraction of C2 at concentrations of 1 to 20 μg/mL ranged from 89.8% to 92.5%, meaning that this fraction of C2 is available to cross tissues.
The blood-plasma partitioning (BP ratio) of C2 in rats was experimentally measured and ranged from 0.6 to 0.71 (Table 6). A BP ratio less than 1 indicates that the compound is free in the plasmatic phase and not inside the blood cells [30].
Discussion
The pharmacological effect of a drug has been described to depend on its pharmacokinetic properties, such as good oral absorption, an appropriate half-life of elimination, good distribution and good bioavailability. The compound C2 (5-[(4-carboxybutanoyl)amino]-2-hydroxybenzoic acid) is a novel synthetic derivative of 5-aminosalicylic acid (5-ASA) that has shown in vitro and ex vivo dual activity as an antioxidant and MPO inhibitor [34–36]. To continue the preclinical study of this compound, we herein reported its pharmacokinetic study in Wistar rats.
For C2 quantification in plasma and other tissues, a RP-HPLC method with UV-Vis detection was first developed, and the validation demonstrated that this method is suitable and complies with the performance parameters established by the FDA and ICH regulations for bioanalytical methods.
In this study, three routes of administration were examined, intragastric, intravenous and intraperitoneal, to delineate the absorption process in the gastrointestinal tract and intraperitoneal tissues. When C2 was administered via the intragastric route, the compound quickly reached the systemic circulation, but unfortunately, the oral bioavailability was only 13% of the administered dose; however, C2 was well distributed in all tissues and able to cross the blood brain barrier. More importantly, the compound reached the small intestine and colon. Therefore, the use of this compound is advantageous to that of 5-ASA for the treatment of UC and CD. Specifically, C2 presented a t1/2 of approximately 33 ± 5 min, and the Vd was 2.0 L/kg. C2 has a short half-life, which may be a disadvantage with respect to 5-ASA for setting the dosing regimen. However, the plasma half-life of salicylates, such as aspirin, is 15 min, and that of salicylate 2–3 h at low doses. Furthermore, C2 was no longer detected in the plasma 6 h after administration, indicating that C2 accumulates in some tissues [44]. This feature may be a disadvantage for toxic compounds, but the acute toxicity study demonstrated that C2 has a low acute toxicity risk. Therefore, the tissue accumulation of C2 may be an advantage, especially for establishing the dosing regimen.
We hypothesize that a fraction of the administered C2 dose is absorbed in the stomach, whereas the remaining fraction reaches the small intestine and colon, where it accumulates for with 6 h or longer. In addition, we associate the low intragastric absorption of C2 with the difficulty to dissolve this compound in isotonic saline solution; therefore, a formulation based on 75% saline (0.9%), 5% Tween 80 and 20% propylene glycol was used according to the proportions permitted for administration in humans [45]. In addition the pH may affect the bioavailability of C2 because this compound is entirely unionized at pH > 3. With respect to intraperitoneal administration, C2 was absorbed at neutral pH despite the fact that it was mostly ionized. However, the hydroxyl group that is attached to the aromatic ring is not ionized, which may promote the absorption of this molecule.
The protein-binding study showed a lower C2 protein binding of 10% than that of 5-ASA (40%), indicating that more C2 would be available to cross tissues and reach the site of action. This phenomenon may arise because drugs are generally weak acids, which bind almost exclusively to the albumin plasma protein that is readily bound to acidic drugs. The binding of drugs to albumin is generally reversible and favored by lipid solubility [46, 47]. Therefore, compounds with low lipo-solubility, such as C2 (Log P = 1.3), are less likely to bind to albumin. Finally, a BP-ration less than 1 indicates that the compounds are free in the plasmatic phase and not inside blood cells, which favors C2 reaching the site of action in a live organism, including humans [42, 43].
Conclusions
In the present study, a bioanalytical method to quantitate C2 (5-[(4-carboxybutanoyl)amino]-2-hydroxybenzoic acid) in rat plasma with an HPLC technique was validated to meet the specified performance parameters for bioanalytical applications provided for the pharmacokinetic evaluation of this new chemical entity. Pharmacokinetic studies showed that the compound is absorbed by the biological membranes and widely distributed in all tissues and that its binding to albumin was minimal.
In summary, we found several important differences and advantages of C2 compared with 5-ASA based on its pharmacokinetic properties in Wistar rats. Considering the pharmacological activity and acute toxicity studies, we propose the use of C2 as a potential candidate to treat inflammatory diseases, including UC and CD.
Acknowledgments
The authors thank the Consejo Nacional de Ciencia y Tecnología (CONACYT: I010/0532/2014; 132353), Instituto de Ciencia y Tecnología del Distrito Federal (ICyTDF/279/2011), Comisión de Operación y Fomento de Actividades Académicas/Secretaría de Investigación y Posgrado-IPN (COFAA-SIP/IPN: 20161383; 20160204; 20161374), and The Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED: 214RT0482) for their financial support. ARC and MGS thank CONACYT for their postdoctoral (290917-IPN) and M. Sc. scholarship (130245), respectively.
Author Contributions
Conceived and designed the experiments: ARC JCB MCRH IIPM JEMW. Performed the experiments: ARC MGS. Analyzed the data: ARC MGS. Contributed reagents/materials/analysis tools: JCB MCRH IIPM JEMW. Wrote the paper: ARC MGS JCB MCRH IIPM JEMW. General design of the research protocol, chromatographic analysis of plasma samples and pharmacokinetic behavior of C2: ARC. Animal treatment, collection and processing of blood and tissue samples, data statistical analysis of the pharmacokinetic behavior of C2: MGS. Design and biological evaluation of C2: JCB MCRH. Contribution in the synthesis and characterization of C2: IIPM. Coordination and execution of research protocol: JEMW.
References
- 1. Cheifetz AS. Management of active Crohn disease. JAMA. 2013;309:2150–8. pmid:23695484
- 2. Singh S, Kullo IJ, Pardi DS, Loftus EV Jr. Epidemiology, risk factors and management of cardiovascular diseases in IBD. Nat Rev Gastroenterol Hepatol. 2015;12:26–35. pmid:25446727
- 3. Dulai PS, Mosli M, Khanna R, Levesque BG, Sandborn WJ, Feagan BG. Vedolizumab for the treatment of moderately to severely active ulcerative colitis. Pharmacotherapy. 2015;35:412–23. pmid:25884529
- 4. O'Toole A, Moss AC. Optimizing biologic agents in ulcerative colitis and Crohn's Disease. Curr Gastroenterol Rep. 2015;17:32. pmid:26188882
- 5. Gasparetto M, Guariso G. Crohn's disease and growth deficiency in children and adolescents. World J Gastroenterol. 2014;20:13219–33. pmid:25309059
- 6. Lee KM, Lee JM. Crohn's disease in Korea: past, present, and future. Korean J Intern Med. 2014;29:558–70. pmid:25228829
- 7. Sales-Campos H, Basso PJ, Alves VB, Fonseca MT, Bonfá G, Nardini V, et al. Classical and recent advances in the treatment of inflammatory bowel diseases. Braz J Med Biol Res. 2015;48:96–107. pmid:25466162
- 8. Burisch J. Crohn's disease and ulcerative colitis. Occurrence, course and prognosis during the first year of disease in a European population-based inception cohort. Dan Med J. 2014;61:B4778. pmid:24393595
- 9. Chaparro M, Gisbert JP. Maintenance therapy options for ulcerative colitis. Expert Opin Pharmacother. 2016;May 30:1–11. [Epub ahead of print].
- 10. Kothari M, Mudireddy P, Swaminath A. Patient considerations in the management of ulcerative colitis—role of vedolizumab. Ther Clin Risk Manag. 2015;19:11:1235–42.
- 11. Iskandar HN, Dhere T, Farraye FA. Ulcerative colitis: update on medical management. Curr Gastroenterol Rep. 2015;17:44. pmid:26386686
- 12. Wang Y, MacDonald JK, Benchimol EI, Griffiths AM, Steinhart AH, Panaccione R, et al. Type I interferons for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2015;9:CD006790. pmid:26368001
- 13. Wei X, Kenny JR, Dickmann L, Maciuca R, Looney C, Tang MT. Assessment of disease related therapeutic protein drug-drug interaction for etrolizumab in patients with moderately to severely active ulcerative colitis. J Clin Pharmacol. 2016;56:693–704. pmid:26412221
- 14. Boyle M, Ting A, Cury DB, Nanda K, Cheifetz AS, Moss A. Adherence to rectal mesalamine in patients with ulcerative colitis. Inflamm Bowel Dis. 2015;21:2873–8. pmid:26313693
- 15. Langan RC, Gotsch PB, Krafczyk MA, Skillinge DD. Ulcerative colitis: diagnosis and treatment. Am Fam Physician. 2007;76:1323–30. pmid:18019875
- 16. Gordon M, Naidoo K, Thomas AG, Akobeng AK. Oral 5-aminosalicylic acid for maintenance of surgically-induced remission in Crohn's disease. Cochrane Database Syst Rev. 2011;19:CD008414.
- 17. Tanida S, Mizoshita T, Ozeki K, Katano T, Kataoka H, Kamiya T, et al. Advances in refractory ulcerative colitis treatment: A new therapeutic target, Annexin A2. World J Gastroenterol. 2015;21:8776–86. pmid:26269667
- 18. Franco AI, Escobar L, García XA, Van Domselaar M, Achecar LM, Luján DR, et al. Mesalazine-induced eosinophilic pneumonia in a patient with ulcerative colitis disease: a case report and literature review. Int J Colorectal Dis. 2016;31:927–9. pmid:26189026
- 19. Alskaf E, Aljoudeh A, Edenborough F. Mesalazine-induced lung fibrosis. BMJ Case Rep. 2013;Apr 9:2013.
- 20. D’Incà R, Paccagnella M, Cardin R, Pathak S, Baldo V, Giron MC, et al. 5-ASA colonic mucosal concentrations resulting from different pharmaceutical formulations in ulcerative colitis. World J Gastroenterol. 2013;19:5665–70. pmid:24039359
- 21. Sandborn WJ, Hanauer SB. The pharmacokinetic profiles of oral mesalazine formulations and mesalazine pro-drugs used in the management of ulcerative colitis Alim Pharm Ther. 2003; 17:29–42.
- 22. Chungi S, Dittert W, Shangel L. Pharmacokinetics of sulfasalazine metabolites in rats following concominant oral administration of riboflavin. Pharm Res. 1989;6:1067–1072. pmid:2576130
- 23. D'Incà R, Paccagnella M, Cardin R, Pathak S, Baldo V, Giron MC, et al. Absorption of 5-aminosalicylic acid from colon and rectum. Brit J Clin Pharmacol. 1988;25:269–72.
- 24. Klotz U. Clinical pharmacokinetics of sulphasalazine, its metabolites and other prodrugs of 5-aminosalicylic acid. Clin Pharmacokinet. 1985;10:285–302. pmid:2864155
- 25. Li W, Zhang ZM, Jiang XL. Once daily versus multiple daily mesalamine therapy for mild to moderate ulcerative colitis: a meta-analysis. Colorectal Dis. 2016;May 23 [Epub ahead of print].
- 26. Ye B, van Langenberg DR. Mesalazine preparations for the treatment of ulcerative colitis: Are all created equal? World J Gastrointest Pharmacol Ther. 2015;6:137–44. pmid:26558148
- 27. Böhm SK, Kruis W. Long-term efficacy and safety of once-daily mesalazine granules for the treatment of active ulcerative colitis. Clin Exp Gastroenterol. 2014;7:369–83. pmid:25285021
- 28. Ham M, Moss AC. Mesalamine in the treatment and maintenance of remission of ulcerative colitis. Exp Rev Clin Pharmacol. 2012;5:113–23.
- 29. Oliveira L, Cohen RD. Maintaining remission in ulcerative colitis—role of once daily extended-release mesalamine. Drug Des Devel Ther. 2011;5:111–6. pmid:21448448
- 30. Parakkal D, Ehrenpreis ED, Thorpe MP, Putt KS, Hannon B. A dynamic model of once-daily 5-aminosalicylic acid predicts clinical efficacy. World J Gastroenterol. 2010;16:136–7. pmid:20039462
- 31. Sonu I, Lin MV, Blonski W, Lichtenstein GR. Clinical pharmacology of 5-ASA compounds in inflammatory bowel disease. Gastroenterol Clin North Am. 2010;39:559–99. pmid:20951918
- 32. Malle E, Furtmüller PG, Sattler W, Obinger C. Myeloperoxidase: a target for new drug development? Br J Pharmacol. 2007;152:838–54. pmid:17592500
- 33. Ramírez Durán LA, Rosales-Hernández MC, Hernández-Rodríguez M, Mendieta-Wejebe JE, Trujillo-Ferrara J, Correa-Basurto J. Mapping myeloperoxidase to identify its promiscuity properties using docking and molecular dynamics simulations. Curr Pharm Des. 2013;19: 2204–15. pmid:23016839
- 34. Gutiérrez Sánchez M, Rosales-Hernández MC, Padilla-Martínez II, Correa-Basurto J, Mendieta-Wejebe JE. In vivo evaluation of two 5-aminosalicylic acid derivatives considered as potential therapeutic agents. FASEB J. 2015;29:Suppl 621.1.
- 35.
Alemán González-Duhart D. Evaluación experimental de derivados arílicos como posibles inhibidores de mieloperoxidasa y estabilizadores de radicales libres, en un modelo de diabetes mellitus tipo 2. Tesis de Maestría en Ciencias en Farmacología. Escuela Superior de Medicina, Instituto Politécnico Nacional. Ciudad de México, México, Agosto 2013.
- 36.
Ramírez Durán LA. Síntesis química y evaluación de compuestos con posible actividad antioxidante e inhibidores de mieloperoxidasa. Tesis de Doctorado en Investigación en Medicina. Escuela Superior de Medicina, Instituto Politécnico Nacional. Ciudad de México, México, Enero 2013.
- 37.
OECD Guidelines for the Testing of Chemicals 425: Acute Oral Toxicity—Up-and-Down-Procedure (UDP). 2008.
- 38.
NOM-062-ZOO-1999. Especificaciones técnicas para la producción, cuidado y uso de los animales de laboratorio, SAGARPA. México. 1999.
- 39.
Morton DB. Humane endpoints in animal experimentation for biomedical research: Ethical, legal and practical aspects. Humane Endpoints in Animal Experiments for Biomedical Research. London: The Royal Society Medical Press; 1999.; pp. 5–12 Available: http://www.lal.org.uk/publications/this-is-a-test-subject/humane-endpoints.
- 40.
Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. FDA. 2001. Available: http://www.fda.gov/cder/guidance/index.html.
- 41.
ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text And Methodology Q2(R1). 2005. Available: http://www.ich.org/products/guidelines/quality/quality-single/article/validation-of-analytical-procedures-text-and-methodology.html.
- 42. Talbi A, Zhao D, Liu Q, Li J, Fan A, Yang W, et al. Pharmacokinetics, tissue distribution, excretion and plasma protein binding studies of wogonin in rats. Molecules. 2014;19:5538–49. pmid:24786691
- 43. Skopp G, Pötsch L, Mauden M, Richter B. Partition coefficient, blood to plasma ratio, protein binding and short-term stability of 11-nor-Δ9-carboxy-tetrahydrocannabinol glucuronide. Forensic Sci Int. 2002;126:17–23. pmid:11955826
- 44.
Pascuzzo-Lima C. Farmacología Básica. ISBN: 978-980-12-3246–9. 2008.
- 45.
Rowe RC, Sheskey PJ, Quinn ME. Handbook of Pharmaceutical Exicipients. Pharmaceutical Press. London-Chicago. 6a Edición. 2009.
- 46. Oravcová J, Böhs B, Lindner W. Drug-protein binding sites. New trends in analytical and experimental methodology. J Chromatogr B Biomed Sci Appl. 1996;677:1–28.
- 47.
Armijo JA. Farmacocinética: Absorción, Distribución y Eliminación de los Fármacos. Ed. Farmacología Humana. Masson. Barcelona. 4a edición. 2003.