Pre-Clinical Compartmental Pharmacokinetic Modeling of 2-[1-hexyloxyethyl]-2-Devinyl Pyropheophorbide-a (HPPH) as a Photosensitizer in Rat Plasma by Validated HPLC Method

Abstract

A second-generation chlorin-based photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) has shown tremendous therapeutic potential in clinical trials in the treatment of esophageal cancer. Herein, we have developed and validated a bioanalytical method for estimation of HPPH in rat plasma using High Performance Liquid Chromatography (HPLC) with a photo diode array (PDA) detector. The method was applied for carrying out pharmacokinetic study of HPPH. Further pharmacokinetic modeling was carried out to understand the compartment kinetics of HPPH. The developed method was fully validated as per the United States Food and Drug Administration (US-FDA) guidelines for bioanalytical method validation. The linearity of the method was in the range of 250–8000 ng/mL, and the plasma recovery was found to be 70%. Pharmacokinetic parameters were evaluated and compared via non-compartment analysis and compartment modeling after the intravenous (i.v.) bolus administration in rats using Phoenix WinNonlin 8.0 (Certara™, U.S.A.). From the obtained results, we hypothesize that the HPPH complies with two compartmental pharmacokinetic model. Furthermore, it was observed that HPPH has the rapid distribution from the central compartment to peripheral compartment along with slow elimination from peripheral compartment.

Graphical Abstract

1. Introduction

There are many methods available for the treatment of cancer, such as surgery, chemotherapy, immunotherapy, radiation therapy, stem cell transplant, photodynamic therapy (PDT) etc. Out of these treatments, PDT has been explored to a largest extent. PDT is a type of treatment that uses photosensitizers or photosensitizing agent and exposure to a specific wavelength of light, which generates reactive oxygen species that kill cancerous cells [1,2]. Initially, the photosensitizing agent is injected into the bloodstream; this agent gets absorbed and deposited in the cells. The photosensitizing agent stays in the cancer cells for a longer period of time compared to the normal cells and then, the tumor is exposed to a specific wavelength of light, thereby activating the agent and generating oxygen to kill the cancer cells [3–5]. Porfimer sodium is the first-generation photosensitizing agent approved by FDA for the treatment of esophageal, non-small cell lung and bladder cancer. However, there are several limitations of this compound, because it is a combination of 60 molecules it is difficult to synthesize, and high concentration of agent along with longer duration of light exposure has to be used for effective treatment [4,5]. HPPH (Fig. 1) is a second-generation chlorin-based photosensitizer. It accumulates in tumor cells and may cause photo allergic skin reactions [6,7]. HPPH has become popular with the brand name Photochlor. Currently it is under phase-II clinical trial for the treatment of esophageal cancer [5].

Fig. 1.

Chemical structures for (I) HPPH (II) Rhodamine-B

Analytical methods that have been reported for the quantification of HPPH, include either radio labeling or the use of hyphenated techniques like LC-MS/MS [8–11]. Bellnier, et al. have developed a method for the estimation of HPPH that utilizes a radio labeled assay and have applied it for determining the pharmacokinetic parameters in mice plasma samples. In these studies they have concluded a long half-life and low plasma clearance of HPPH [8]. However, radio labeled HPPH and near-infrared reflectance imaging is not an appropriate method for estimating HPPH in plasma samples [10]. Radio labeling may change the pharmacokinetic parameters, which leads to decreased accuracy of the results. In addition, radioactive agents can be toxic to humans and the environment [12]. Chen, et al. have reported different methods for the quantification of HPPH in serum samples using HPLC coupled with a fluorescence detector, LC–MS/MS and fluorescence-based microplate reader for conducting clinical pharmacokinetic studies, in which they have reported long half-life and very low clearance of HPPH [11]. However, LC-MS/MS has been established as a powerful technique for the quantitative determination of analytes in biological samples. Unfortunately, it requires high maintenance cost and needs trained personnel, which hampers the routine analysis. Pharmacokinetic studies utilizing compartment modeling has yet to be explored for HPPH. Therefore, to overcome all the above hurdles, a simple, sensitive, robust method was developed for estimation of HPPH in plasma.

The aim of the present work was to develop and validate HPPH in plasma and to explore the possible compartmental pharmacokinetics of HPPH. The bioanalytical method was validated using Rhodamine-B (RDB) as an internal standard (I.S) (Fig. 1). Extensive sample clean-up techniques are often required to suppress elevated baseline noise due to co-extracted interferences. Sample clean-up processes for biological matrices often rely on solid phase extraction (SPE) techniques, which include, conditioning, sample loading, washing, sample elution and evaporative reconstitution steps that are performed via our previously reported method [13]. Full validation has been performed including system suitability, selectivity, limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, dilution integrity, carry over effect and stability studies. All the bioanalytical method validation parameters were evaluated as per US FDA guidelines. The stability studies were also investigated for HPPH in plasma samples after storage in various environments including bench top, autosampler, freeze-thaw cycles, short-term and long-term studies. Compartmental pharmacokinetics of HPPH was explored by using the developed method in Wistar rats after administration of 4 mg/kg through intravenous route [14]. Evaluation of pharmacokinetic parameters were performed by non-compartmental analysis and compartment modeling based on the data obtained from concentration time profile by using Phoenix WinNolin 8.0 (Certera™, U.S.A.) software.

2. Materials and Methods

2.1. Chemicals, Materials and reagents

HPPH was synthesized in-house (Chemical Biology Laboratory, NCI-Frederick, NIH Frederick, MD, U.S.A). RDB was procured from Sisco Research Laboratories Pvt. Ltd. (SRL) (Mumbai, India). HPLC grade acetonitrile (ACN) and methanol were procured from Merck Limited (Mumbai, India). Ammonium formate and formic acid were procured from Sigma Aldrich (St. Louis, MO, USA). SPE cartridges, Cleanert PEP-3 SPE were procured from Agela Technologies (Wilmington, USA). Milli-Q water was produced in-house using from a Milli-Q water purification system (Millipore, USA). All the other chemicals and solvents were of analytical grade and were used as such without any further purification. Wistar rats (female) weighing around 200–220g were purchased from Central Animal Facility of Birla Institute of Technology & Science, Pilani, India. Rats were housed under proper cages as per standard guidelines. All experimental procedures were performed as per CPCSEA guidelines and approved (Protocol approval # IAEC/RES/20/09) by local Institutional Animal Ethics Committee of BITS Pilani, Pilani campus.

2.2. Instrumental and analytical conditions

The Liquid Chromatography was carried out on a Shimadzu prominence HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump (LC-10AT), Photo diode array (PDA) detector (SPD-M20A), Column oven (CTO-10AS) and auto sampler (SIL-HTA, Shimadzu, Kyoto, Japan). Data acquisition and controlling the system was ascertained by LC solution software (Version 1.24 SP1). Chromatographic separation was achieved at 40 ± 0.5°C using a Durashell C8 (250 mm x 4.6 mm) column (Agela Technologies, Wilmington, USA). The binary mobile phase consisted of ACN and ammonium formate (pH 4.2; 0.01M) (92:8 %v/v) in an isocratic mode at a flow rate of 1 mL/min, degassed by vacuum filtration prior to use. The detections were carried out with injection volume of 50 μL at a wavelength of 406 nm.

2.3. Solution Preparations: Calibration standards (CS) and quality control (QC) standards

HPPH stock was prepared by dissolving an accurately weighed amount in ACN, to obtain a final concentration of 1 mg/mL. A working stock solution of HPPH was prepared by diluting with ACN to make 500 μg/mL from stock solution of HPPH (1 mg/mL). From working stock solution (500 μg/mL), working standard solutions of 5, 10, 30, 60, 100, 130 and 160 μg/mL were prepared. The working QC solutions were prepared as 8, 80 and 140 μg/mL. Further spiking was carried out in plasma by utilizing working standard solutions to result in concentrations of 250, 500, 1500, 3000, 5000, 6500 and 8000 ng/mL. The QC standards were also prepared separately in the same manner at three concentration levels, such as low QC (LQC, 400 ng/mL), medium QC (MQC, 4000 ng/mL and high QC (HQC, 7000 ng/mL). All the stock and working standard samples were stored at −20°C till further analysis.

2.4. Sample Preparation

The extraction technique utilized for sample processing plays a vital role in terms of sensitivity, selectivity and recovery. To optimize the sample preparation, we have executed different extraction techniques like protein precipitation, liquid-liquid extraction and solid phase extraction (SPE). However, SPE process has showed its significant advantage in terms of consistent recovery and selectivity of the analyte. A 300 μL aliquot of rat plasma with HPPH was taken in 2 mL capacity of polypropylene disposable tube in which 20 μL of 100 μg/mL of RDB (I.S) solution (I.S solution was prepared in ACN) was added and were vortexed for 1 min. Then, 300 μL of ammonium formate buffer was added and vortexed for 1 min on a vortex mixer. The samples were then loaded into the SPE cartridges. The sequential steps involved in SPE procedure is similar to our previous published reports with slight modifications as mentioned in supplementary data; Table S1 [13]. The SPE eluents were evaporated to dryness under nitrogen at 45 °C. The dried residue was reconstituted in 100 μL mobile phase, vortexed and sonicated for 30 sec each. Finally, 50 μL extracted sample was injected through autoinjector for detection [15–17].

2.5. Assay validation procedures

The bioanalytical method was validated as per USFDA guidelines for bioanalytical method validation [18,19].

2.5.1. System suitability

System suitability is typically a system performance test, which was carried out by injecting the same concentration sample by six times for checking the column efficiency, reproductivity and resolution. It was performed to verify the holistic function of the chromatography system on a day-to-day basis.

2.5.2. Selectivity

In this method, there was no interference observed at the retention time of analyte and IS. For performing the selectivity, plasma samples from different Wistar rats were collected and were analyzed as per the developed method.

2.5.3. Linearity

Linearity was assessed with a concentration range between 250 ng/mL to 8000 ng/mL using seven calibration standards. Six different plasma calibration curves standards were prepared by plotting peak area ratios of each analyte to IS versus nominal plasma concentrations. The data obtained were fitted by linear regression with the selected weighing factor. The selection was carried out after utilizing various weighing factors (1/var, 1/x, 1/x², 1/$\sqrt{x}$, 1/y, 1/y², 1/$\sqrt{y}$), and weighing factor was selected based on the percentage relative error (% RE).

2.5.4. Limit of detection (LOD) and limit of quantitation (LOQ)

The limit of detection (LOD) is defined as the lowest concentration of analytes that can be detected and differentiated from the noise level.

The limit of quantitation (LOQ) is defined as the lowest concentration of analytes that can be quantified with accepted accuracy and precision recommendations (not exceeding ±20%).

Signal to noise (S/N) ratio was used for estimation of LOD and LOQ. First the signal to noise ratio was checked for standard samples at system suitability step. LOD and LOQ were calculated based on equation 1. Desired S/N values for LOD was 3 and for LOQ was 10.

$$\text{Concentration of LOQ and LOD}=\frac{\text{Concentration of Standard}}{\frac{\text{S}}{\text{N}}\text{value of Standard}}\text{ X Desired}\frac{\text{S}}{\text{N}}\text{value}$$ eq.1

Generally, LOQ is the first point in CC and analyzed plasma samples by spiking with LLOQ concentration in six replicates (n=6).

2.5.5. Precision and accuracy

The precision and accuracy for intra-day and inter-day assay were obtained by analyzing QC samples (LQC, MQC, HQC) and LLOQ. The intra-day assay precision and accuracy were determined in six replicates by analyzing samples on same day, while inter-day assay precision and accuracy were determined in six replicates by analyzing each QC and LLOQ samples on three consecutive days. Measured concentrations were calculated by comparison with a freshly prepared calibration curve. Precision and accuracy, ≤±15% were considered as acceptable, except at the LLOQ which should not deviate more than ±20%.

2.5.6. Recovery

The percentage recovery was assessed by assaying plasma samples spiked with HPPH at LLOQ, LQC, MQC and HQC levels (n=3) and was compared with the same spiked HPPH concentration in mobile phase.

2.5.7. Dilution integrity and Carry over effect

Blank plasma was spiked with 2 times higher than the ULOQ concentration and was diluted 5 times and 10 times with blank plasma (n=6) to obtain a concentration of 3200 ng/mL and 1600 ng/mL for dilution integrity. Diluted samples were assayed alongside a freshly prepared calibration curve. The accuracy and precision values should be within ±15%.

Carry over effects were assessed in blank plasma after injecting upper limit of quantification calibration standard (ULOQ, 8000 ng/mL). As per the guidelines the sample dilutions must be performed to ensure the plasma sample dilution integrity [20].

2.5.8. Stability studies

Stability of the analyte in the plasma matrix was investigated by subjecting them to different storage conditions in various environments. The stability of stock solution was assessed for 30 days at −20 ±05 °C and working standard solutions for 48 h at 4 ±0.5 °C.

Stability studies were conducted for freeze–thaw for three cycles at −80 ±05 °C, bench top at ambient temperature, autosampler conditions, short-term storage for 15 days at −80 ±05 °C and long-term storage for 3 months at −80 ±05 °C. Stability studies were performed in replicates of three at different QC levels (LQC, MQC, and HQC) and at LLOQ. All stability samples were compared against freshly spiked calibration curves.

2.5.9. Ruggedness

Ruggedness was established by using the different column of same chemistry and company and analysis was performed by a second analyst. Accuracy and precision at QC levels and LLOQ (n=3) was checked.

2.6. Pharmacokinetic study in Wistar rats

To investigate the developed assay method, pharmacokinetic study of HPPH was performed on young Wistar rats weighing around 200–220 g. HPPH was dissolved in 10% tween 80 and 5% DMSO in normal saline (4 mg/mL). 4 mg/kg of HPPH was administered intravenously to each rat (n=4). Blood samples were collected in 1.5 mL capacity of polypropylene disposable tubes at 5, 15 and 45 min, 2, 4, 6, 12, 24, 48, 72, 100, 120, 144, 168, 192 and 250 h. Blood samples were centrifuged at 6000 rpm for 15 min and plasma samples were kept in −80 ±10 °C until analysis. The plasma concentrations time profile of HPPH was plotted to generate pharmacokinetic parameters.

Pharmacokinetic parameters were estimated using Phoenix WinNolin8.0 (Certera™, U.S.A) with both non-compartmental and compartmental modeling. Akaike’s information criterion (AIC), Schwarz. Bayesian Criteria (SBC) and linear regression coefficient were used to select the finest set of pharmacokinetic models (single, two or multi compartment). The lowest AIC and SBC values was preferred to estimate the pharmacokinetic model [21].

3. Results and Discussion

3.1. Method Development

3.1.1. Optimization of chromatographic parameters and sample preparation technique

Method development strategies were followed to obtain the optimum parameters that was both simple and economical. A methodological approach was followed during method development, mobile phase selection (pH, polarity), selection of stationary phase, flow rate, column oven temperature and injection volume were finalized for obtaining peak symmetry, consistent recovery and optimum resolution between the analyte and IS. Initially organic modifiers (methanol and ACN) were used with varying concentrations of buffers (ammonium acetate, sodium acetate and ammonium formate) in different ratios. After successive optimization, ACN-ammonium formate (pH:4.2; 0.01 M) was selected as mobile phase which gave symmetrical peak shape and good peak intensities. HPPH is a highly hydrophobic molecule, we tried different reversed phase C18 columns such as Waters, Agela technologies and Phenomenex for detection. Unfortunately, all of the columns showed longer retention time (more than 20 min) and peak broadening. This was mainly due to stronger affinity of reversed phase C18 columns with highly hydrophobic molecule. Therefore, we selected C8 column to get better resolution, selectivity and retention time (fast analysis). Durashell C8column (250×4.6 mm, 5μ) column with ACN-ammonium formate (pH:4.2; 0.01M) mobile phase in ratio of 92:8 % v/v at a flow rate of 1 mL/min was finally selected for analysis of the analyte and IS. The injection volume was 50 μL and optimized retention time for HPPH and RDB (IS) were 11.84 and 7.98 min, respectively. The overall run time was 15 min and detection wavelength selected as 406 nm.

Plasma sample preparation technique is widely used to get the cleaner sample without interference of endogenous substances with maximum recovery. Different sample preparation techniques were tried, initially, sample pre-treatment was done by protein precipitation using ACN and methanol which gave poor and inconsistent recovery. Then, liquid-liquid extraction was performed by using methylene chloride, hexane and ethyl acetate still it produced less recovery with high back ground noise. Finally, we preferred SPE method for extraction process. SPE provided much cleaner sample extracts, less solvent consumption, higher recoveries and shows proper reproducibility (supplementary data; Table S2).

3.2. Assay validation

3.2.1. System Suitability and Selectivity

By performing the system suitability test, we conformed that system was working fine and it showed reproducibility and proper resolution.

The sample clean-up procedure was more selective because there is no significant plasma matrix interference at retention time of analyte. The chromatograms of blank plasma, (Plasma sample without analytes), LLOQ plasma sample (250 ng/mL), plasma sample showing only analyte (HPPH) and plasma sample showing only IS (RDB) is shown in Fig. 2.

Fig. 2.

HPLC Chromatograms for (A) Blank plasma (B) Only IS (C) Only HPPH (D) LOQ (250ng/mL) (E) LOD (80ng/mL) (F) Calibration standard

3.2.2. Linearity, LOD and LLOQ

The calibration standard curve showed reproducibility over the selected concentration range of 250–8000 ng/mL with R² values more than 0.9999. The R² values, slopes and intercepts were calculated from five intra and inter day calibration curves. The optimized weighed linear regression model was 1/x² which showed minimum percentage relative error (%RE) presented in supplementary data; Table S3. The accuracy (%RE) and precision (%CV) of the method has been represented in Table 1.

Table: 1.

Precision and accuracy data of back calculated concentrations of calibration standard samples of HPPH in rat plasma (n = 5)

Nominal concentration (ng/mL)	Measured concentration (Mean ± SD, ng/mL)	Precision (% CV)	Accuracy (% bias)
250	263.091 ± 21.700	8.248	5.236
500	485.455 ± 18.624	3.834	−2.857
1500	1473.300 ± 54.325	3.734	−2.998
3000	2959.089 ± 56.432	1.882	−0.029
5000	4986.839 ± 59.017	1.173	0.594
6500	6376.916 ± 37.768	0.592	−1.894
8000	8301.850 ± 36.002	0.434	3.773

SD, standard deviation; CV, coefficient of variation

In this method, LOD for HPPH was 80 ng/mL which was based on S/N ratio ≥ 3. The LLOQ obtained for the method was 250 ng/mL which was selected on the basis of S/N ratio ≥ 10. Moreover, LLOQ (250 ng/mL) was quantified accurately and precisely (± 20).

3.2.3. Precision and accuracy

The deviation in inter and intra-day precision at three QC samples (LQC, MQC, HQC) and LLOQ was ≤ 8.76 and ≤ 8.75 being represented in Table 2. Whereas, accuracy (% bias) was found to be in the range of −3.70 to 12.23. Based on the values, it was conformed that the variations were within the acceptable limits as per recommended guidelines.

Table: 2.

Precision (% CV) and accuracy (% bias) of HPPH in rat plasma samples at QC levels (n=5).

		Inter-day			Intra-day
Level	Nominal Conc. (ng/mL)	Measured Conc. (Mean ± SD, ng/mL)	Precision (% CV)	Accuracy (% bias)	Measured Conc. (Mean ± SD, ng/mL)	Precision (% CV)	Accuracy (% bias)
LLOQ	250	245.697 ± 9.994	8.769	−1.721	280.582 ± 13.033	8.759	12.233
LQC	400	418.648 ± 13.605	1.534	1.865	362.907 ± 16.571	1.994	−3.709
MQC	4000	4014.408 ± 17.336	0.446	0.360	4002.597 ± 10.600	0.274	0.065
HQC	7000	6828.619 ± 13.174	0.197	−2.448	7028.474 ± 18.655	0.270	0.407

3.2.4. Recovery, Dilution integrity and Carry over

The percent recoveries of HPPH under optimized procedure for all three QC samples and LLOQ are listed in Table 3. The percentage mean recovery of HPPH was found to be ≤ 65.49%. The results of dilution integrity performed were within acceptable limits. Results were obtained after 5 times and 10 times dilution with blank plasma (n=6). The accuracy value after 5 times and 10 times dilution were found to 98.75 ±2.84 and 100.64 ±3.57% respectively. Further no carry over effect was seen.

Table: 3.

Absolute recoveries (%) of HPPH in rat plasma samples from QC levels

			Recovery (%)
Level	Nominal concentration (ng/mL)	n	Mean ± SD (n=3)	% CV
LLOQ	250	3	62.50 ± 7.22	6.82
LQC	400	3	63.31 ± 5.95	5.10
MQC	4000	3	65.49 ± 9.21	3.12
HQC	7000	3	65.28 ± 2.69	2.96

n, number of samples; SD, standard deviation; CV, coefficient of variation

3.2.5. Stability studies

The stability studies for HPPH were performed as per the procedure described in section 2.5.8. HPPH was found to be stable under varied stability conditions. Results conforms that HPPH was stable under different storage conditions, that HPPH may be subjected under normal processing and storage conditions (Table 4).

Table: 4.

Stability of HPPH in rat plasma at QC levels

Stability	Nominal concentration (ng/mL)	Measured Concentration (ng/mL) ± SD	Precision (% CV)	Accuracy (% bias)
0 h (for all)	250	245.69 ± 9.99	8.769	−1.721
	400	418.64 ± 13.60	1.534	1.865
	4000	4014.40 ± 17.33	0.446	0.446
	7000	6828.61 ± 13.17	0.197	0.197
Autosampler (48 h)	250	252.61 ± 9.50	3.764	1.045
	400	399.97 ± 10.89	2.724	−0.007
	4000	4024.67 ± 43.62	1.083	0.616
	7000	7097.39 ± 53.22	0.749	1.391
Bench-top (24 h, RT)	250	268.18 ± 14.88	5.549	7.275
	400	395.85 ± 25.49	6.439	−1.036
	4000	3989.07 ± 60.16	1.510	−0.448
	7000	7018.01 ± 56.12	0.799	0.257
Freeze-thaw (−80 °C,	250	244.77 ± 6.88	2.814	−1.629
3 cycle)	400	423.08 ± 21.44	5.069	4.872
	4000	4044.84 ± 58.36	1.443	1.590
	7000	7076.22 ± 39.83	0.562	1.007
Short-term (−80 °C,15 days)	250	232.46 ± 5.35	2.302	−7.013
	400	369.33 ± 5.50	1.491	−7.666
	4000	4069.00 ± 34.07	0.837	1.725
	7000	6974.00 ± 28.79	0.412	−0.371
Long-term (−80 °C, 3 months)	250	245.80 ± 15.46	6.293	−1.680
	400	372.33 ± 13.57	3.646	−6.916
	4000	3972.33 ± 79.14	1.992	−0.691
	7000	6870.67 ± 27.06	0.393	−1.847

3.2.6. Ruggedness

The accuracy and precision of all three QC samples and LLOQ performed by second analyst by using a different column of same company and specifications were ≤ −2.44 and +8.76% respectively. Variation of accuracy in calibration standards was ≤ 2.99% for all the concentrations of HPPH.

3.3. Pharmacokinetic study in Wistar rats

To investigate the developed assay method, pharmacokinetic study of HPPH was performed on young Wistar rats. To explore the possible compartmental pharmacokinetic models, HPPH was administered intravenously at a dose of 4 mg/kg. For determining the pharmacokinetic profile precisely, selection of proper compartmental model (one or two) is important. AIC, SBC and coefficient correlation (R²) values suggests the suitable criterion for the selection of pharmacokinetic compartment model. The smaller AIC and SBC values indicate the proper fit to the concentration-time profile [21]. The mean AIC, SBC and R² values for one compartmental model were 373.77, 375.95 and 0.88, respectively. The mean AIC, SBC and R² values for two compartmental model were 319.67, 324.04 and 0.987, respectively. Herein, an appropriate and validated mathematical compartmental pharmacokinetic modeling of HPPH was developed. Compartmental pharmacokinetic modeling of predicted and observed drug concentrations-time profile is represented in Fig. 3, no bias was observed in between predicted and observed concentrations in case of two-compartment model. From these results, we hypothesize that the HPPH complies with two compartmental model (supplementary data; Fig. S1). The generated data suggested that the distribution and elimination are the two different distribution processes which are responsible for the above outcome. The equation for the two-compartmental model is Cp_(t) = A e^−αt + B e^−βt. The pharmacokinetic parameters α and β are the distribution and elimination rate constants, whereas A and B values are the intercepts for distribution and elimination phases. Shorter distribution half-life (t_1/2α) of HPPH, might be due to rapid distribution from the central compartment to peripheral compartment. However, HPPH showed a longer elimination half-life (t_1/2β), due to slow distribution and elimination from peripheral compartment [22]. Other pharmacokinetic parameters, such as area under the concentration time curve (AUC), the initial drug concentration (C₀), clearance (Cl) were simulated by using non-compartmental and compartmental modeling. All the pharmacokinetic parameters of HPPH are represented in the Table 5. Mean ± SEM plasma concentration versus time profile of HPPH is shown in Fig. 4.

Fig. 3.

Compartmental pharmacokinetic modeling of predicted and observed drug concentrations-time profile (A) one-compartment model (B) two-compartment model

Table: 5.

The non-compartmental and compartmental pharmacokinetic parameters for HPPH after i.v. bolus (4 mg/kg) administration to rat (n = 4)

		HPPH (4 mg/kg; i.v.)
Pharmacokinetic Parameters	Units	NC	1-C	2-C
C 0	ng/ml	7923.78 ± 141.63	--	---
AUC0-t	h*ng/ml	205135.9 ± 949.90	95553.25 ± 320.82	218314.01 ± 133.95
AUC0-α	h*ng/ml	217137.6 ± 976.53	---	---
AUMC	h*h*ng/ml	18466052 ± 725.74	2431570.01 ± 150.72	19334868.01 ± 250.45
MRT	h	85.09 ± 2.21	21.86 ± 10.81	88.29 ± 6.96
Kel	1/h	0.011 ± 0.01	---	---
T1/2	h	63.81 ± 3.75	---	---
T1/2α	h	---	---	1.07 ± 0.79
T1/2β	h	---	---	62.90 ± 6.62
CL	ml/h/kg	18.46 ± 0.86	49.10 ± 21.58	18.39 ± 1.14
Vd	ml/kg	1695.41 ± 151.67	---	---
V1	ml/kg	---	844.57 ± 166.05	597.02 ± 95.58
V2	ml/kg	---		1022.15 ± 139.99
K10	1/h	---	0.071 ± 0.032	0.039 ± 0.026
K12	1/h	---	---	0.901 ± 0.106
K21	1/h	---	---	0.301 ± 0.051
R2	---	---	0.8807 ± 0.04	0.9878 ± 0.01
AIC	---	---	373.77 ± 20.61	319.68 ± 10.21
SBC	---	---	375.95 ± 20.61	324.04 ± 10.21

NC: non-compartmental; 1-C: one-compartmental; 2-C: two-compartmental; AIC: Akaike information criterion; SBC: Schwarz-Bayes Criterion

Fig. 4.

(A) Mean plasma concentration-time profile of HPPH (Mean ± SD, n = 4) (B) Representative chromatogram of plasma sample collected after 1 h of drug administration

4. Conclusion

HPPH is a lipophilic, second-generation, chlorin-based photosensitizer, used in photodynamic therapy. The aim of the present study was to develop a bioanalytical assay method for quantitation of HPPH in plasma matrix and to explore its compartmental pharmacokinetics. The developed method is simple, sensitive, and economical with high through put utilizing very less organic solvents, hence environment friendly. Moreover, we have carried out full validation of this method, stability studies and compartmental pharmacokinetics modeling. To the best of our knowledge, this method has been applied for the first-time to explore detailed compartmental pharmacokinetics profile of HPPH. Our constructive findings offer complete information regarding compartmental pharmacokinetics of HPPH.