Method of Determining Pyrrole–Imidazole Polyamide in Rat Plasma Using Ultra-Fast Liquid Chromatography-Ultraviolet Spectrometry

Abstract

To improve the efficiency of drug-discovery research on pyrrole–imidazole polyamides (PIs), a more rapid method for quantitative and qualitative measurement of PI in rat plasma samples was developed here using ultra-fast liquid chromatography-ultraviolet spectrometry (UFLC-UV) in order to shorten the measurement time. A measurement method of PIs by HPLC developed until now takes 45 min for one sample measurement. This method was inefficient to investigate extraction conditions from biological samples and measurement of animal experimental samples. In the developed method of this study, PI and phenacetin (internal standard, IS) were separated with an ACQUITY UPLC HSS T3 (1.8 µm, 2.1 × 50 mm; Nihon Waters K.K., Japan) column using a mobile phase of 0.1% acetic acid (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.3 mL/min with a linear gradient. The detection wavelength was 310 nm. The calibration curve was linear in the range of 0.225–4.5 µg/mL (correlation coefficients ≥0.9995, n = 5). The intra- and inter-day accuracies were in the range of −6.04 to 12.2%, and the precision was less than 2.99%. The measurement time of this method (7 min per injection) was markedly shortened to about one-sixth of the previous measurement time (45 min per injection). This is the first report describing the quantitative and qualitative measurement of PI in plasma using UFLC-UV. The present method will be very useful for the drug-discovery research of PIs.

INTRODUCTION

Pyrrole–imidazole polyamides (PIs) are DNA recognized peptide that were initially identified from antibiotics such as duocarmycin A and distamycin A. They recognize and bind DNA with specific sequences and are composed of the aromatic rings of N-methylpyrrole and N-methylimidazole amino acids.^1–3) A chemically synthesized PI was found to have a strong affinity to a minor groove of sequence-specific double-helical DNA.¹⁾ In accordance with that property, various types of DNA-binding PIs have been developed to regulate gene expression by targeting the promoter regions of enhancer and transcription factor binding elements in vitro.^1–4) Dickinson et al.⁵⁾ reported that the genes of human immunodeficiency virus (HIV) were silenced by PIs that bind specifically to their regulatory sequences. It was reported that the expression of transforming growth factor β1 (TGF-β1) mRNA and protein in the renal cortex of Dahl-S rats was significantly inhibited by a PI.⁶⁾ Urinary protein and albumin in Dahl-S rats were also reduced by the PI and the reduction was independent of changes in blood pressure.⁶⁾ Igarashi et al.⁷⁾ also reported a preclinical study of a PI (GB 1101) that targets the human TGF-β1 gene as a transcriptional gene silencer for hypertrophic scars in a common marmoset primate model. These findings indicate that PI targeting TGF-β1 should be a novel gene-silencing agent for TGF-β1-associated diseases, including progressive renal diseases and hypertrophic scarring after surgical operations and skin burns.^6,7) As described above, it is anticipated that PIs can be used as novel drugs for gene therapy and PIs are expected to be put into practical use.

We previously reported the methods of detecting other PIs using high-performance liquid chromatography-ultraviolet spectrophotometry (HPLC-UV) and high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).^8–11) Generally, HPLC-UV requires larger amounts of sample and longer analysis time compare to LC-MS/MS. The shorter analysis time for a sample by LC-MS/MS (5.5 min)¹⁰⁾ is a greater advantage than that of the HPLC-UV (15 and 45 min),^8,9,11) however, the installation of LC-MS/MS could be cost-prohibitive for many research facilities including authors.’ Thus, to promote fundamental research efficiently with lower cost, it is necessary to improve on the currently available methods. In order to improve the current analysis capacity by HPLC-UV with a limited budget, we investigated efficient and precise conditions for measuring PI using an ultra-fast liquid chromatography (UFLC)-UV method. In this study, UFLC-UV method was focused to develop a cost effective and reasonably rapid method in a practical range to look for a happy medium.

This is the first research on the analysis method for PIs applying UFLC-UV, so for as we know. We attempted to improve the method of measuring PI (GB 1101) by focusing on TGF-β1 here. Among various PIs, GB1101 was regarded as the most promising drug.^6,7)

MATERIALS AND METHODS

Reagents and Solutions

PI was synthesized by Nihon University School of Medicine following the method of Bando et al.¹²⁾ The chemical structure of PI was shown in Fig. 1. Acetic acid was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Phenacetin was purchased from Sigma-Aldrich Co. LLC (Tokyo, Japan) and used as an internal standard (IS). Acetonitrile and methanol were all of HPLC-grade, and were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Water was obtained from a water purification system (Direct-Q; Nihon Millipore Ltd., Tokyo, Japan). Sprague-Dawley rat plasma was purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan).

Fig. 1. Chemical Structure of PI (GB1101, MW: 1787.90)

Chromatography Conditions and Instruments

The UFLC-UV system consisted of a Prominence UFLC (20 A Series) (Shimadzu Co., Kyoto, Japan). PI and IS were analyzed on an ACQUITY UPLC HSS T3 (1.8 µm, 2.1 × 50 mm; Nihon Waters K.K., Tokyo, Japan). The column and autosampler were maintained at 40 and 4°C, respectively. Mobile phases A (0.1% acetic acid) and B (acetonitrile) were used for examining the separation between PI and IS, at a flow rate of 0.3 mL/min. The injection volume was 10 µL and the detection wavelength was set to 310 nm. These conditions were used in reference to previously published papers.^8–11)

Preparation of Standard Samples

A primary stock solution of PI (1 mg/mL) was prepared by dissolving the compounds in 0.1% acetic acid. The solutions were stored at 4°C. Secondary stock solutions were prepared by diluting the primary stock solution with 0.1% acetic acid just before use. The solutions for calibration standard samples were prepared at concentrations of 5, 10, 15, 20, and 25 µg/mL, and those for rat plasma calibration curve samples were made at concentrations of 2.25, 3, 7.5, 15, 22.5, 30, 37.5, and 45 µg/mL. An IS was dissolved in methanol (0.5 mM).

Preparation of Samples for Investigating Separation Conditions of UFLC

Samples for examining the separation conditions were as follows: The same volumes of secondary standard solution of 5 µg/mL PI and 1 mM IS were mixed (PI sample). Moreover, 1 mM IS was mixed with the same volume of 0.1% acetic acid (IS sample). A blank sample was prepared by mixing the same amounts of 0.1% acetic acid and methanol.

Preparation of Rat Plasma Samples for Calibration Curve

Calibration standard plasma samples were prepared by mixing 5 µL of secondary stock solutions and 45 µL of blank plasma (final concentrations of PI: 0.225, 0.3, 0.75, 1.5, 2.25, 3, 3.75, and 4.5 µg/mL), to create a rat plasma calibration curve.

Pretreatment of Rat Plasma Samples

The rat plasma samples for calibration curves and the plasma from PI-administered rats were pretreated with methanol. The plasma samples of 50 µL were mixed with 100 µL of methanol containing 0.5 mM IS and vortexed. After centrifugation at 10000 × g for 5 min at 4°C, the supernatant was obtained. The centrifugation was repeated twice and the supernatant (10 µL) was injected into the UFLC system.

Animals and Drug Administration

Two male 12-week-old Sprague-Dawley rats weighed 344 and 345 g were purchased from Japan SLC, Inc. (Tokyo, Japan). A cannula was inserted into the right femoral artery and right jugular vein of each rat by the vendor. The experiment was performed at least 1 d after purchase. The rats were housed in a temperature-controlled room under a 12-h light–dark cycle and were allowed free access to food and water. PI in 0.1% acetic acid (6.0 mg/kg) was administered in a single intravenous dose. The sampling times were 0, 0.16, 0.33, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h after administration. Each sample was immediately transferred to a heparinized microcentrifuge tube. These samples were separated by centrifugation at 4°C and 10000 × g for 10 min and stored at −80°C until use. After sampling, the collected blood was replaced with an equal volume of saline. The plasma concentration–time profiles of PI were analyzed by a non-compartmental method. The area under the plasma concentration–time curve (AUC) and the area under the first moment curve (AUMC) were obtained using the linear trapezoidal rule and extrapolated to infinity. The terminal elimination rate constant (k_e) was calculated by regression of the terminal log-linear portion of the plasma concentration curve. The terminal elimination half-life (t_1/2) was calculated to be 0.693/k_e. The clearance (CL), mean residence time (MRT), and the volume of distribution in the steady state (V_ss) were calculated as dose/AUC, AUMC/AUC, and CL*MRT, respectively. The plasma concentrations of PI were extrapolated to time zero (C₀). Nihon University Animal Care and Use Committee (Tokyo, Japan) approved the animal experiment.

Method Validation

Evaluation of the method was performed as follows applying the procedures previously reported.¹³⁾ The method was validated for selectivity, recovery, linearity, accuracy, and precision. The validation was conducted in accordance with the guidelines of the National Institute of Health Sciences (NIHS).¹⁴⁾ The selectivity was evaluated by examining the separation of PI and IS from the plasma matrix components of blank rat plasma. The recovery was determined for the intra-day (n = 3) and inter-day (n = 3) precision at a concentration of 3 µg/mL PI. The recovery was determined by comparing the absolute peak areas of the extracted samples with those of the pre-spiked standards. The calibration curves were constructed by plotting the PI peak area divided by the IS peak area. The linearity of the calibration curves was evaluated by linear regression analysis. The lower limit of quantitation (LLOQ) of PI was experimentally defined as the lowest concentration of the calibration curve that could be measured with acceptable accuracy and precision.

The intra- and inter-day accuracy and precision of this method were investigated using working solutions of PI. Accuracy was expressed as a percentage of the measured concentration relative to the theoretical concentration.

  

The criterion for acceptable accuracy was defined as a mean concentration within ±15% of the nominal concentration, except for the case of LLOQ (0.225 µg/mL), for which it should not exceed ±20%.¹⁴⁾ Precision was expressed as the relative standard deviation (RSD). The acceptance criterion for precision was defined as the RSD at each concentration not exceeding 15%, except for the case of LLOQ, for which it should not exceed 20%.¹⁴⁾

RESULTS AND DISCUSSION

Investigation of Gradient Elution

At the first onset, a mobile phase for UFLC analysis were investigated using isocratic elution and LC-MS/MS gradient elution reported by Nagashima et al.¹⁰⁾

Various isocratic elutions were investigated, however, an appropriate condition to avoid peak splitting, tailing, broadening, and peak overlapping with the blank peak could not be find. Applying the LC-MS/MS gradient elution for UFLC without any modification resulted PI peak overlapping with the IS peak. It was assumed that the PI and IS overlapping was happened during non-polar environment, acetonitrile : 0.1% acetic acid = 95 : 5. Considering the results, various gradient patters were studied based on the LC-MS/MS gradient pattern paying attention on non-polar condition.

Table 1 summarizes the investigated conditions of the representative gradient mobile phase of eluent B. The initial gradient time influenced the retention time of PI and IS. When this time was prolonged, the retention times of both compounds increased (data not shown). The next isocratic eluent ratio and time affected the separation of PI and IS. It was possible to separate the peaks of PI, IS, and contaminants in all gradient patterns. To select the optimal conditions from the conditions in Table 1, a calibration curve was prepared under each condition, and the correlation coefficient and the accuracy and precision were calculated.

Table 1. Gradient Pattern of UFLC for PI and IS Separation

Pattern		0 min	0.5 min	1 min	4 min	4.01 min	7 min
I	B, %	0		60	60	0	0
II	B, %	0		50	50	0	0
III	B, %	0	40		40	0	0
IV	B, %	5		50	50	5	5

Tables 2 and 3 show the obtained results. The results of accuracy, precision, and correlation coefficient at gradient pattern III were not as good as those of gradient patterns I, II, and IV. The degrees of separation differed slightly among gradient patterns I, II, and IV. Gradient pattern IV, in which the change rate of the eluent was low, was adopted as the optimal condition in order to shorten the time of return of the eluent to its initial state after measurement. The total measurement time of this gradient was 7 min per injection (including the time of return to the initial conditions for the next measurement).

Table 2. Intra-Day Accuracy and Precision of Each Gradient Pattern

	Nominal concentration (µg/mL)	Mean concentration (µg/mL)	Accuracy (%)	Precision (%)
Pattern I	5	4.998	3.41	−0.04
	10	10.17	1.04	1.69
	15	14.78	0.13	−1.50
	20	19.95	1.58	−0.25
	25	25.11	0.43	0.43
Pattern II	5	4.93	3.70	−1.50
	10	10.22	1.44	2.21
	15	14.85	0.71	−0.97
	20	19.93	1.87	−0.33
	25	25.07	0.53	0.28
Pattern III	5	5.25	4.32	4.96
	10	11.11	1.54	11.1
	15	12.91	25.0	−13.9
	20	19.49	4.14	−2.55
	25	26.06	2.36	4.23
Pattern IV	5	4.96	1.09	−0.77
	10	9.93	1.94	−0.75
	15	15.23	2.35	1.53
	20	19.92	1.93	−0.40
	25	24.96	1.28	−0.14

Table 3. Calibration Curve Parameters for Each Gradient Pattern (n = 3)

	Slope	Intercept	Correlation coefficient
Pattern I	0.829 ± 0.012	0.132 ± 0.137	0.9998
Pattern II	0.827 ± 0.011	0.229 ± 0.113	0.9998
Pattern III	0.788 ± 0.021	0.167 ± 0.591	0.9863
Pattern IV	1.377 ± 0.019	0.303 ± 0.349	0.9998

Results of Rat Plasma Sample Validation

For validation, recovery, linearity of the calibration curves, accuracy, and precision were investigated. Table 4 shows the recovery of 3 µg/mL PI and 0.5 mM IS from rat plasma. Both compounds were recovered at high yield. The linearity of the calibration curves of PI was measured in the range of 0.225 to 4.5 µg/mL in the rat plasma (n = 5). Chromatograms of blank rat plasma and blank rat plasma spiked with PI (4.5 µg/mL) under the optimal condition presented in Table 1 pattern IV are shown in Fig. 2. The relationships between the PI peak area divided by the IS peak area and the corresponding concentrations were found to be linear. The correlation coefficients were ≥0.9995. Table 5 shows the results of accuracy and precision. The intra- and inter-day accuracy for all concentrations was within the range of −6.04 to 12.2%. In addition, the intra- and inter-day precision was less than 2.99%. These values complied with NIHS guidelines.

Fig. 2. UFLC Chromatograms of a Blank Rat Plasma Sample (Lower) and Blank Rat Plasma Spiked with PI (Upper)

Chromatogram is measurement results using optimal gradient pattern IV in Table 1. Peak 1: 4.5 µg/mL PI, Peak 2: 0.5 mM IS.

Table 4. Recovery of PI and IS in Rat Plasma Samples

	Recovery (%)
	Intra-day (n = 3)	Inter-day (n = 3)
PI	98.19 ± 3.10	96.67 ± 4.69
IS	102.2 ± 6.53	110.2 ± 7.07

Table 5. Intra- and Inter-day Accuracy and Precision in Rat Plasma Samples

Theoretical concentration (µg/mL)	Intra-day (n = 5)			Inter-day (n = 3)
	Mean concentration (µg/mL)	Accuracy (%)	Precision (%)	Mean concentration (µg/mL)	Accuracy (%)	Precision (%)
0.225	0.23	2.60	2.51	0.25	12.2	0.64
0.300	0.29	−3.04	1.47	0.30	0.55	0.50
0.750	0.73	−2.25	2.09	0.70	−6.04	0.55
1.500	1.47	−1.81	2.99	1.45	−3.29	1.38
2.250	2.31	2.69	2.22	2.32	2.98	0.54
3.000	3.02	0.53	0.52	3.04	1.17	0.36
3.750	3.75	−0.08	1.48	3.74	−0.25	0.50
4.500	4.47	−0.58	0.42	4.47	−0.57	0.11

Application to Animal Experiments

Chromatograms of rat plasma collected at zero and 0.16 h after administration of PI are shown in Fig. 3. Figure 4 shows the plasma concentration versus time plots for PI. The validated UFLC-UV method was successfully applied to determine PI in a PK study. Table 6 lists the PK parameters of PI in rats.

Fig. 3. Chromatograms of Rat Plasma Collected at Zero (Lower) and 0.16 h (Upper) after Administration of PI after Intravenous Administration (6.0 mg/kg)

Chromatogram is measurement results using optimal gradient pattern IV in Table 1. Peak 1: PI, Peak 2: 0.5 mM IS.

Fig. 4. Rat Plasma Concentration–Time Profile after Intravenous Administration (6.0 mg/kg)

PI in 0.1% acetic acid (6.0 mg/kg) was administered a single intravenous dose to two male 12-week-old Sprague-Dawley rats. The sampling times were 0, 0.16, 0.33, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h after the administration.

Table 6. The Pharmacokinetic Parameters of PI in Rats after Single Intravenous Dose of PI at 6.0 mg/kg (n = 2)

Pharmacokinetic parameters	Rat 1	Rat 2
C0 (µg/mL)	2.45	2.49
ke (1/h)	0.27	0.34
t1/2 (h)	2.59	2.06
CL (mL/h)	629.4	310.9
Vss (mL)	2351.5	924.2
AUC0–∞ (µg h/mL)	3.28	6.65

C₀: initial concentration k_e: elimination rate constant t_1/2: half-life of PI elimination at the terminal phase CL: clearance V_ss: volume of distribution in the steady state AUC_0–∞: area under the plasma concentration–time curve from 0 h to infinity.

CONCLUSION

In this research, we aimed to establish a method for measuring PI using UFLC-UV. In the measurement method studied here, it was possible to shorten the measurement time from 45¹¹⁾ to 7 min. To analyze all samples (n = 123) in Tables 4 and 5, it takes 92.25 h (3.84 d) by HPLC, compared with 14.35 h by the UFLC method reported in this paper (not including time for sample preparation and pretreatment). Via this improvement, we can expect to boost the efficiency of research and reduce costs. In the range of 0.225 to 4.5 µg/mL, applicable results were obtained in terms of linearity, accuracy, and accuracy of the calibration curve in daily fluctuations. When rat plasma was used as a biological sample, it was revealed that the methods developed in this research can quantify PI with good accuracy and reproducibility. In addition, the detection sensitivity was improved about fourfold. The minimum quantification limit by UFLC-UV in rat plasma was reduced to 0.225 µg/mL. Though the type of PI was different, the lower limit of quantifications were 1 or 0.25 µg/mL (HPLC-UV)^8,9) and 10 ng/mL (LC-MS/MS).¹⁰⁾ The assay sensitivity was slightly improved compared to HPLC-UV. The measurement method newly developed in this investigation is useful for measuring blood concentrations of various PIs in pharmaceutical research.

Acknowledgments

This work was supported by JSPS Grant-in-Aid for Scientific Research (C) Grant Number 17K09716. The authors thank Fumiya Murakami, BSc, Tamako Kaminoyama, BSc, Hidetada Koyama, BSc, Asako Muranami, BSc, Ayumi Nagamine, BSc, and Yuka Satou, BSc, for their assistance.

Conflict of Interest

The authors declare no conflict of interest.

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