Synthesis, Characterization, and In Vivo Evaluation of Desmethyl Anethole Trithione Phosphate Prodrug for Ameliorating Cerebral Ischemia-Reperfusion Injury in Rats

Abstract

Anethol trithione (ATT) has a wide range of physiological activities, but its use is limited due to its poor water solubility. To improve the solubility of ATT, we synthesized and characterized a novel phosphate prodrug (ATXP) relying on the availability of the hydroxy group in 5-(4-hydroxyphenyl)-3H-1,2-dithiole3-thione (ATX), which was transformed from ATT rapidly and extensively in vivo. Our results showed that ATXP significantly improved drug solubility. ATXP was rapidly converted to ATX and reached a maximum plasma concentration with a T_max of approximately 5 min after intravenous (iv) administration. Furthermore, after the oral administration of ATXP, the C_max was 3326.30 ± 566.50 ng/mL, which was approximately 5-fold greater than that of the parent drug form, indicating that ATXP has greater absorption than that of ATT. Additionally, the oral phosphate prodrug ATXP increased the ATX in the area under the plasma concentration vs time curves (AUC_0–t = 3927.40 ± 321.50 and AUC_0–∞ = 4579.0 ± 756.30), making its use in practical applications more meaningful. Finally, compared to the vehicle, ATXP was confirmed to maintain the bioactivity of the parent drug for a significant reduction in infarct volume 24 h after reperfusion. Based on these findings, the phosphate prodrug ATXP is a potentially useful water-soluble prodrug with improved pharmacokinetic properties.

1. Introduction

Anethol trithione (ATT) (Figure 1) is a sulfur heterocyclic compound that has attracted attention due to its function as a slow-releasing H₂S donor.¹ Additionally, ATT possesses various pharmacological properties. It has been used for decades for treating xerostomia by enhancing salivary secretion and increasing muscarinic acetylcholine receptors.² It is a bile secretion-stimulating drug or cholagogue that protects the liver via an increase in glutathione levels and phase II detoxifying enzymes.³ ATT is a potentially efficacious chemoprevention agent for lung cancer and exerts chemopreventive effects in several target organs, such as the liver and colon, by increasing the detoxification rate of carcinogens in these target organs.⁴⁻⁶ In addition, ATT protects blood–brain barrier integrity following cerebral ischemia, an effect largely mediated by significantly elevated serum H₂S released by ATT.⁷ However, the use of ATT is greatly restricted by its poor water solubility (0.38 μg/mL) and limited oral bioavailability in vivo because of its extreme lipophilicity.^8,9

Figure 1.

Synthetic protocol of ATXP phosphate prodrugs.

Some pharmaceutical methods have been used to improve the hydrophilicity and oral bioavailability of ATT. Lipid-based formulations have been explored to enhance the oral bioavailability of the poorly water-soluble drug ATT, and the AUC_0–8h and C_max from the lipid formulations are 1.4- to 3.0-fold and 2.0- to 4.8-fold higher than those from suspensions, respectively.⁹ However, lipidic vehicles might influence CYP-mediated metabolism in the liver; hence, the proportion of ATT metabolized to 4-hydroxy-anethole trithione (ATX) was significantly reduced.¹⁰ Furthermore, water-soluble analogs of ATT were synthesized by substituting the methyl group of ATT with the simple hydrophilic alkylamino group, and the amine salt prodrugs have a solubility of up to 20 mg/mL.¹¹ The conversion of this amine salt prodrug to ATX via O-demethylation by O-demethylase was consistent with ATT metabolism.^11,12 However, the activity of O-demethylase varies between different populations, and reduced activity has been found in patients with impaired liver function, which probably causes differences in the conversion of the above prodrugs in different populations.^13,14 This finding encouraged us to apply prodrug design methods based on ATT’s active metabolite (ATX). However, ATX also has poor solubility. To improve the solubility of ATX, the methyl group was substituted with various lipid groups via ester linkage with the hydroxyl group of ATX, but the solubility remained limited, only 660.6 ng/mL, which was approximately 14 times that of ATT.¹⁵ Therefore, it is necessary to find a new method to greatly improve the solubility of ATT while simultaneously avoiding any adverse side effects on the human body.

Phosphate ester prodrug strategies are widely used for improving aqueous solubility for oral administration, especially for drugs intended for parenteral administration.¹⁶ The active parent drug molecule is rapidly released from phosphate prodrugs by endogenous phosphatases, such as alkaline phosphatase, which is expressed in many tissues throughout the body.¹⁷ The solubility of the phosphate sodium salt of the acacetin prodrug was more than 1.9 million timeshigher than that of the parent drug acacetin, which would be suitable for developing an intravenous (iv) preparation.¹⁸ UNIL088 is a water-soluble prodrug of cyclosporine A (CsA) developed for topical eye delivery.¹⁹ These phosphate prodrugs have significantly greater water solubility than their parent drugs, thereby enhancing absorption, bioavailability, and patient acceptance.

Based on previous studies, it is desirable to improve the solubility of ATT by using an ATX phosphate precursor. ATX has a hydroxyl group that provides a place for intervention by phosphate precursors, thereby controlling the properties of ATX. Notably, ATT has important physiological activities, such as protecting the rat brain from focal cerebral ischemia-reperfusion (IR) injury.⁷ Therefore, whether ATX prodrugs retain these activities is worthy of attention and research.

2. Results and Discussion

2.1. Solubility

ATT has extremely low water solubility (0.38 μg/mL).^9,10 After an oral therapeutic dose, the plasma concentration of ATT becomes very low. This low plasma concentration is observed because ATT is metabolized rapidly in vivo into ATX via O-demethylation.^10,12 Although the solubility of ATX was better than that of ATT, its solubility remained very low (Table 1) and did not meet the requirements of iv administration. In the present study, ATT was not directly used as a parent drug for prodrug design; rather, its active metabolite ATX was used for prodrug synthesis. As mentioned in the literature review, ATT is quickly metabolized into ATX via O-demethylation by O-demethylase, which has a similar pharmacological activity to ATT.^11,12 Therefore, it can be assumed that ATX is the active form of ATT. Prodrug design strategies are based on the active metabolites, which can reduce the difference in drug metabolism due to differences in the expression and activity of individual O-demethylase enzymes.^13,14,20 Furthermore, the presence of the hydroxyl group of ATX provides a handle for phosphate prodrug intervention, whereby solubility can be improved. Thus, this study aimed to assess the importance of the phosphate prodrug ATXP in improving water solubility and retaining ATT activity. As expected, the aqueous solubility of ATT was greatly enhanced by the prodrug ATXP because this compound has a solubility of approximately 2.1 mg/mL in ultrapure water, 1.5 mg/mL in NaCl solution (pH = 7.4), and 1.7 mg/mL in phosphate-buffered saline (PBS) (pH = 7.4) (Table 1). Thus, the prodrug ATXP increases ATX and ATT solubility by over 1800-fold, which could allow drug dissolution in saline solution at concentrations useful for therapeutic applications.

Table 1. Aqueous Solubilities of ATXP and ATX in Buffer Solution, the Half-Lives of ATXP in Saline Solution and the Half-Life of ATXP in Alkaline Phosphatase Solution (Mean ± SD, n = At Least 3).

compound		solubilitya (mg/mL)	solubilityb (mg/mL)	solubilityc (mg/mL)	chemical stability, t1/2	enzymatic hydrolysis, t1/2
ATXP	1 h	2.0755 ± 0.09230	1.5026 ± 0.14922	1.7546 ± 0.15901	stableb	<5 s
	24 h	1.9378 ± 0.15365	1.5620 ± 0.14840	1.7938 ± 0.01604
	48 h	2.1804 ± 0.00767	1.5432 ± 0.00445	1.6900 ± 0.01478
ATX	1 h	0.0008 ± 0.00002	d	d	e	e

^aUltrapure water.

^b0.9% NaCl solution, pH = 7.4.

^cPBS, pH = 7.4.

^dBelow the detection limit.

^eNot determined.

2.2. Stability in Saline Solution

The stability of the prodrug was assessed in saline solution at 37 °C. Experiments showed that ATXP was highly stable, and the parent drug ATX was not detected after incubation for 12 h in saline solution, indicating that ATXP did not undergo spontaneous hydrolysis. ATXP remained stable even after 30 days of continuous incubation in saline solution (Figure 2A). The stability of ATXP was an important foundation for the development of a drug used for iv application. The above results also suggest that ATX released from ATXP in plasma in the next experiment in the present study was not due to poor stability but to hydrolysis of ATXP by phosphatase.

Figure 2.

ATXP can be hydrolyzed by alkaline phosphatase in vitro. The concentration of HP was determined by high-performance liquid chromatography (HPLC). (A) Stability of the prodrug was examined in saline solution at 37 °C and sampled at 0 h, 12 h, 24 h, 3 days, and 30 days. Then, HPLC was applied to determine the amount of ATX released in the ATXP saline solution. (B) ATX can be released rapidly from ATXP by alkaline phosphatase and detected by HPLC. (C) ATXP (300 μM) was hydrolyzed by alkaline phosphatase at pH 7.4, 37 °C with a half-life of less than 5 s. (D) ATXP (80 μM) was hydrolyzed in human plasma at 37 °C, and samples were determined at different time points.

2.3. Hydrolysis of ATXP in Alkaline Phosphatase Solution and Plasma

Evaluation of the alkaline phosphatase hydrolysis of ATXP was carried out in a boric acid buffer solution at pH 7.4 and 37 °C. ATXP was completely hydrolyzed into ATX in less than 5 s (Figure 2B,C). The hydrolysis study revealed that ATX can be effectively hydrolyzed by alkaline phosphatase, which is promising for further drug development. In our previous study, the half-life (t_1/2) value of the phosphate prodrug of honokiol was 8.9 s, and it was completely released by hydrolysis at approximately 20 s.²¹ This faster process may be the result of fewer phosphate ester groups in ATXP. ATX has only one hydroxyl group to form an ester with a phosphate group, whereas honokiol has two groups that can undergo this reaction. This result also suggests the possibility for prolonged hydrolysis time via increasing the number of phosphate esters and for pharmacokinetic regulation.

However, the alkaline phosphatase used in this experiment was derived from the bovine intestinal mucosa. Due to the differences in species, we used plasma to determine whether ATXP can be hydrolyzed to ATX by phosphatase in plasma. As expected, the prodrug ATXP released ATX in human plasma, but this hydrolysis process was milder than the above enzyme buffer system and took nearly 24 h, with 25 μM ATX released from ATXP (Figure 2D). The activity of alkaline phosphatase in plasma for these substrates is obviously lower than that in many tissues; alkaline phosphatase activity is especially abundant in hepatic, skeletal, and renal tissues.^17,22 Thus, unsurprisingly, the hydrolysis rate of ATXP was relatively moderate in plasma. Moreover, the complex components in plasma, such as plasma proteins, affect the hydrolysis of prodrugs. For example, the extremely slow hydrolysis rate of a prodrug can be induced by the high plasma protein binding of the prodrug.²³ In contrast, prodrugs showing low protein binding have increased the availability of prodrugs for hydrolysis in plasma.²⁴ Taken together, these findings strongly suggest that the in vivo administration of the prodrug ATXP could rapidly release the parent drug. Therefore, it was necessary to conduct pharmacokinetic experiments with the prodrug ATXP.

2.4. Pharmacokinetics of ATXP and ATT in Rats

The fast-enzymatic hydrolysis of ATXP suggested that the prodrug could be transformed into the active compound ATX promptly to exert its biological activity in vivo. This quick hydrolysis was also reflected in the pharmacokinetic parameters, such as T_max. Our pharmacokinetics study demonstrated that after iv administration, ATXP was rapidly converted to ATX with a T_max of approximately 5 min (Table 2). Although there was a slow transformation of the prodrug ATXP in the plasma incubation experiment in vitro (Figure 2C), once the drug was administered in vivo, the hydrolysis process for the prodrug ATPX was greatly accelerated due to the abundance of alkaline phosphatase in tissues.

Table 2. Noncompartmental Pharmacokinetic Parameters of ATX in Rat Plasma Samples from Three Groups (ATXP-IV Group, ATXP-PO Group, and ATT-PO Group) after Intravenous or Oral Administration.

variable	ATXP-IV-12.5 (mg/kg)	ATXP-PO-25 (mg/kg)	ATT-PO-17.1 (mg/kg)
t1/2 (h)	2.40 ± 1.01	3.44 ± 1.63	6.60 ± 1.92
Tmax (h)	0.08 ± 0.00	0.30 ± 0.00	0.52 ± 0.00
Cmax (ng/mL)	28 259.33 ± 7317.95	3326.30 ± 566.50	709.77 ± 222.93
AUC(0–t) (h·ng/mL)	9033.82 ± 1911.90	3927.40 ± 321.50	2364.49 ± 216.11
AUC(0–∞) (h·ng/mL)	9183.78 ± 1946.18	4579.0 ± 756.30	4193.51 ± 784.91
Vz (mL/kg)	4778.37 ± 2184.37
CL [mL/(h kg)]	1399.19 ± 270.33
MRT(0–t) (h)	0.47 ± 0.12	3.09 ± 0.04	4.46 ± 0.57
MRT(0–∞) (h)	0.66 ± 0.08	3.09 ± 0.04	11.60 ± 2.95

The effect of solubility optimization is one of the key elements of pharmacokinetic enhancement.²⁵ The prodrug approach is a promising molecular modification by which drug developers and designers can modulate drug pharmacokinetics.²⁶ This idea encouraged us to determine whether the pharmacokinetics were improved after the oral administration of ATXP. As expected, our pharmacokinetic study demonstrated that after the oral administration of 25 mg/kg ATXP (equimolar with 17.1 mg/kg ATT), the C_max of the ATXP-PO group was 3326.30 ± 566.50 ng/mL, which was approximately 5-fold higher than that of the ATT-PO group (709.77 ± 222.93 ng/mL, Table 2). Compared to the parent drug form, the oral phosphate prodrug AXTP indeed resulted in improved absorption. This finding also agrees with our earlier observations, which showed that the C_max of the oral phosphate prodrug group was 10 times that of the parent drug PO group.²¹

Additionally, the ATXP-PO group (Figure 3B) showed a greater increase in ATX in the area under the plasma concentration vs time curves (AUC_(0–t) = 3927.40 ± 321.50 h·ng/mL) than did the ATT-PO group (AUC_(0–t) = 2364.49 ± 216.11 h·ng/mL). Furthermore, the T_max (0.30 h) of the ATXP-PO group was markedly shorter than that of the ATT-PO group (0.52 h). In contrast, the t_1/2 of ATX in the ATT-PO group (6.60 ± 1.92 h) was longer than that in the ATXP-PO group (3.44 ± 1.63 h), reflecting the slow absorption of ATT after PO dosing in the digestive tract. Moreover, we concluded that the prolonged mean residence time (MRT) of the ATT-PO group [MRT_(0–∞) = 11.60 ± 2.95] was greater than that of the phosphate prodrug ATXP group [MRT_(0–∞) = 3.09 ± 0.04].

Figure 3.

Concentration of ATX was determined by liquid chromatography–mass spectrometry (LC–MS) after intravenous and oral administration with equimolar amounts of ATXP and ATT in the plasma of rats in vivo at several points. (A) Mean plasma concentration–time curves of ATX after intravenous administration (ATXP-15 mg/kg); (B) mean plasma concentration–time curves of ATX after oral administration (ATXP-25 mg/kg, ATT-17.1 mg/kg). Each point represents the mean ± SD (n = 3).

Our findings were inconsistent with those of previous studies, which suggested that compared to the parent drugs, oral phosphate prodrugs could hardly improve the absorption because the rapid parent drug generation via intestinal alkaline phosphatase led to parent drug precipitation.²⁷ A possible explanation from a previous study for this issue involves an increased absorptive flux across the intestinal mucosa due to intraluminal supersaturation of the parent drug released from intestinal dephosphorylation of the prodrug.²⁸ This inconsistency may also be because ATX is directly released from the prodrug in the intestine, while ATX in the plasma of the oral ATT administration group was converted from O-demethylation by O-demethylase in the liver after ATT absorption in the intestine. Based on these findings, the phosphate prodrug design method for ATT was successfully verified and ATXP was readily converted to ATX in vivo in rats for iv or per os (po) administration with improved pharmacokinetic properties. Hence, whether the prodrugs retained these pharmacological activities deserves further research.

2.5. ATXP Ameliorated IR Injury in Middle Cerebral Artery Occlusion (MCAO) Rats

According to previous studies, ATT plays protective roles during brain IR injury in rats by protecting against damage to the blood–brain barrier through H₂S released from inorganic ATT.⁷ Due to the insolubility of ATT, it has been administered in a vehicle composed of 10% dimethylsulfoxide (DMSO) in corn oil.⁷ However, this vehicle cannot be used in actual clinical practice. In the present study, the phosphate prodrug of ATX exhibits great optimization of the solubility parameters and effective release of ATX. Therefore, it was necessary to confirm whether ATX could maintain its bioactivity to ameliorate IR injury after structural transformation.

In the present study, edaravone, a low-molecular-weight antioxidant drug targeting peroxyl radicals among many types of reactive oxygen species, was chosen as a positive drug control.²⁹ Because of its capacity to scavenge free radicals, edaravone iv infusion has been approved in Japan and China as a drug to treat acute ischemic stroke.^29,30 In addition, ATXP and edaravone are freely soluble in saline (0.9%), while ATT has limited use due to its poor water solubility. Therefore, we did not establish an ATT experimental group. After 2 h of blood flow interruption and 15 min of reperfusion, certain degrees of edema and damage occurred in the brain tissue of rats. Compared to rats that received a vehicle, rats that received ATXP (5 mg/kg) or edaravone (6 mg/kg) showed significant reductions in infarct volume at 24 h after reperfusion (P = 0.0020 and 0.0489, respectively), as shown in Figure 4A,B. There was no significant difference between the ATXP group and the edaravone group (P > 0.05). In addition, the neurological deficits were determined in all rats in the present study. Our results showed varying degrees of deficit, with damaged sensorimotor ability in the vehicle group and the protective role of ATXP and edaravone in reducing the neurological deficit score (Figure 4C). In detail, all groups immediately exhibited an obvious neurological damage phenotype after reperfusion (0 h), but only the neurological deficit score of the vehicle group increased after 24 h of reperfusion (P = 0.0379), reflecting that the neurological deficits were further aggravated without the drug intervention. In contrast, the ATXP- and edaravone-treated groups did not exhibit increased neurological scores after 24 h of reperfusion (P = 0.671 and 0.4298, respectively), indicating the inhibition of neurological damage by the treatment strategy. Our results suggested that ATXP could indeed protect against IR-induced brain damage and inhibit the further aggravation of neurological impairment, consistent with the results of a previous study on ATT.⁷

Figure 4.

ATXP attenuated focal cerebral I/R injury. (A) Quantification of infarct volumes 24 h after focal ischemia. (B) Representative images of TTC staining. (C) Quantification of neurological Bederson scores of ATXP, edaravone and vehicle-treated rats at 0 and 24 h after MCAO. Bars represent the mean ± standard error of the mean (SEM).

Stroke is a leading cause of morbidity and mortality, and timely treatment and intervention can minimize long-term disability by salvaging the at-risk penumbra and consequently reducing the associated morbidity and mortality, which requires the rapid onset of drugs.³¹ Hence, ATXP could be used to treat stroke via iv injection owing to its great water solubility to achieve a rapid onset. These results indicated that ATXP might be a potent neuroprotective agent against focal cerebral IR injury.

3. Conclusions

Our phosphate prodrug design focused on using ATT active metabolites (ATX) as the parent drug and masking the hydroxy group of ATX. As the phosphate prodrug, ATXP greatly enhanced the aqueous solubility of ATX or ATT. ATXP can readily release ATX in rats after both iv and po administration. ATXP retained its desired biological activity of ameliorating IR injury after structural transformation. In conclusion, the phosphate prodrug ATXP is a potentially useful water-soluble prodrug with improved pharmacokinetic properties that could merit further development as a drug candidate.

4. Materials and Methods

4.1. Materials

ATT was purchased from J&K Scientific Ltd. (Beijing, China). Alkaline phosphatase was obtained from Sigma-Aldrich (St. Louis, MO). Phosphorus oxychloride and other reagents were obtained from Chengdu Kelong Chemical Company (Chengdu, China).

4.2. ATX

ATX was synthesized according to a modified version of a previously described procedure.¹¹ As shown in Figure 1, ATT (20.00 g, 83.20 mmol) and anhydrous pyridine hydrochloride (57.80 g, 0.50 mol) were added to a round-bottomed flask. The mixture was heated to 220 °C for 30 min under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, and 200 mL ethyl acetate and 200 mL water were added and stirred for 30 min at 50 °C. The suspension was filtered; next, the organic layer was separated, washed three times with water, dried with Na₂SO₄, and then decolorized with activated carbon overnight. The mixture was filtrated, the filtrate was concentrated until solid precipitation occurred, and 50 mL petroleum ether was added to the residue and then crystallized to provide ATX as an orange solid (12.00 g, 64%; Figures S1–S3 Supporting Information). The purity (>95%) was determined by HPLC (Table S4 and Figure S8, Supporting Information).

4.3. ATX Sodium Phosphate (ATXP)

As shown in Figure 1, ATX (10 g) was dissolved in 200 mL dichloromethane, and pyridine (21 g) was added into the ATX dichloromethane solution. This mixture was lowered to a temperature below −10 °C. Then, phosphorus oxychloride (27 g) was added dropwise to the solution. The reaction mixture was stirred for approximately 5 h at −5 to 0 °C until ATX completely disappeared. After the reaction was completed, ice water (200 mL) was added, and the mixture was stirred for another 30 min. The compound (ATX phosphate) was deposited at the bottom of the bottle as an oil. The supernatant fraction was dumped, and the residue was washed twice with 1 N HCl (40 mL). The crude product was dissolved in 100 mL tetrahydrofuran and dried with Na₂SO₄, filtered and concentrated to provide the compound (ATX phosphate) (12.00 g, 88.6%). For the ATX phosphate (10 g) solution in ethanol (30 mL), the saturated sodium methoxide solution in methanol was added dropwise at room temperature until neutral pH conditions were achieved. The mixture was stirred for 30 min at 25 °C, filtered, and dried to obtain the crude (ATXP) brown solid (9.17 g, 80.0%). Crude ATXP (9.17 g) was dissolved in H₂O (44 mL) at 70 °C and then cooled to room temperature slowly. Anhydrous ethanol (88 g, 111 mL) was added to the above solution (in this process, an amount of product precipitated). The suspension was stirred for 1 h, filtered, and dried to provide the ATXP light red solid (disodium salt of ATX phosphate, 5.83 g, 64%; Figures S4–S6, Supporting Information). The purity (>95%) was determined by HPLC (Table S3 and Figure S7, Supporting Information).

4.4. High-Performance Liquid Chromatography (HPLC)

HPLC analysis was performed on Waters 2695 apparatus coupled to Waters 2996 on a photodiode array detector and analyzed by Waters Empower 2 software (Waters, Milford, MA). HPLC was performed using a Kro-masil 100-5 C18 column (5 μm, 250 mm × 4.6 mm), a mobile phase of 75% methanol, and ultrapure water containing 1% trifluoroacetic acid (TFA) at a flow rate of 1 mL/min and a detection wavelength set at 350 nm.

4.5. Detection of ATXP and ATX Solubility

The aqueous solubilities of ATXP and ATX were determined at 25 °C in ultrapure water, 0.9% NaCl solution (pH = 7.4), and PBS (pH = 7.4), respectively. Excess amounts of ATXP and ATX were added to 1.5 mL polypropylene centrifuge microtubes containing 1 mL of the above solution. The mixtures were incubated for 1, 24, and 48 h, sampled, filtered (0.22 μm, Millipore, Billerca, MA), and analyzed by HPLC. The experiments were repeated three times.

4.6. Stability of ATXP in Saline

ATXP was dissolved in 0.9% sodium chloride solution (0.9% NaCl solution, pH = 7.4) at 65 μg/mL and aliquoted after sterilization via filtration. The prepared ATXP solution was incubated at 37 °C and sampled at 0, 12, 24 h, 3, and 30 days. Then, HPLC was applied to determine the amount of ATX released in the ATXP saline solution, which reflects the stability of ATXP in saline. The experiments were performed in triplicate.

4.7. Hydrolysis of ATXP by Alkaline Phosphatase

To determine whether ATXP can be hydrolyzed in alkaline phosphatase, ATXP was dissolved in a boric acid buffer solution at pH 7.4 (final concentration, 300 μM, in triplicate). After sterilization by filtration, the vehicle control group and experimental group were simultaneously preheated at 37 °C for 15 min. Then, excess alkaline phosphatase was added to the above groups (5000 DEA units/L) and sampled at 2, 5, 10, 15, 30, 60, 600 s, and 30 min. Finally, HPLC was used to detect the amount of ATX released from ATXP by alkaline phosphatase.

4.8. Hydrolysis of ATXP in Human Plasma

The blood donors were healthy individuals who provided informed consent. Lithium heparin blood samples were centrifuged at 2000g for 5 min to remove cells and platelets, and the plasma was collected. Subsequently, ATXP was added to the plasma (final concentration, 80 μM) and incubated at 37 °C for 0, 0.5, 1, 2, 4, 8, 12, and 24 h. The plasma sample (200 μL) was mixed with precooled ethyl acetate for 5 min in 1.5 mL polypropylene centrifuge tubes, and then the mixture was centrifuged at 6000g for 10 min. The clear supernatant was analyzed using HPLC by measuring the concentration of ATX, which was released from ATXP. Data are from representative experiments in triplicate.

4.9. Pharmacokinetics Study

Male Sprague-Dawley rats (7 weeks old, each weighing 220–250 g) were purchased from Beijing WeiTong Lihua Experimental Animal Technology company (Beijing, China). Rats were divided into three groups (n = 3 animals per group): ATXP intravenous treatment group (ATXP-IV group, 12.5 mg/kg), ATXP oral treatment group (ATXP-PO group, 25 mg/kg), and ATT oral treatment group (ATT-PO group, 17.1 mg/kg). Rats in the ATXP-PO group and ATT-PO group were orally administered an equimolar mass of ATX. For intravenous injection, the ATXP was dissolved in saline (0.1% w/w polysorbate 80, pH = 7.0–7.4) at a concentration of 2.5 mg/mL, and the injection amount was 5 mL/kg. For oral administration, ATT was dissolved in 10% hydroxypropyl-β-cyclodextrin (HPβCD) to obtain a concentration of 1.71 mg/mL, and the gavage volume was 10 mL/kg. ATXP was dissolved in saline water to obtain a solution with a concentration of 2.5 mg/mL (0.1% w/w polysorbate 80), and the gavage volume was 10 mL/kg. Blood samples were collected through a cannulated tube at designated times of 0, 5, 15, 30 min, 1, 2, 4, 6, 8, and 24 h into tubes containing heparin sodium. Plasma was separated from the samples by centrifugation (2000g, 6 min, 2–8 °C; plasma samples were stored at −80 °C before analysis). Finally, an aliquot of 200 μL of the supernatant was analyzed by LC–MS/MS (Tables S1 and S2, Supporting Information).

4.10. Establishment of the Middle Cerebral Artery Occlusion (MCAO) Model

The ethics committee of Chengdu Medical College approved the experimental program, and all animal experiments were conducted in accordance with the Chinese Animal Welfare Law. Eight-week-old male Sprague-Dawley rats weighing approximately 280 g (obtained from Beijing Weitong Lihua Experimental Animal Technology company, Beijing, China) were anesthetized with halothane (3%) and subjected to MCAO as previously described, with minor modifications of the method established by EZ Longa.³² In brief, a 2 cm incision was made along the ventral midline, and the shallow fascial muscle tissue of the right neck was carefully dissected. The external, internal, and common carotid arteries (external carotid artery (ECA), internal carotid artery (ICA), and common carotid artery (CCA), respectively) were exposed in turn. Clamping the ICA and then ligating the ECA makes it easier to mobilize the CCA. The distal end of the CCA was tied, and a suture was placed around the CCA at the proximal end to ensure that the blood vessels were not blocked. Arteriotomy was performed between the two lines on the CCA. A 4-0 monofilament nylon suture was inserted in the ICA ∼18 mm into the ECA incision site. Insertion of the suture was halted when slight resistance was encountered. At this point, the suture blocked the blood flow of the right MCA. Ischemic rats were placed in an environment at 37 °C for 2 h; then, the sutures were carefully removed to allow MCA reperfusion.

Rats were randomly divided into three groups (n = 12 animals per group), and the following drugs were intravenously administered: the control group was treated with tail vein injection of 0.9% saline, the positive control group was treated with a single dose of 6 mg/kg edaravone immediately after reperfusion by tail vein injection, and the experimental group was treated with a double dose of 5 mg/kg ATXP. The first dose of ATXP was administered immediately after reperfusion, and the second dose was given 5–6 h later. The control group was treated with physiological saline, which was administered in the same manner as ATXP.

4.11. Measurement of Infarct Volume

After the treatment, the rats were euthanized. The skull cavity was carefully removed to expose the entire brain. The brain was immediately placed in a freezer at −20 °C until it was completely frozen. The frozen brain tissue of each rat was cut into 2-mm-thick sections for a total of six slices. Then, the slices were placed in 5% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO) and stained at 37 °C for 15–20 min in the dark. Brain slices were removed for immersion in a 4% formaldehyde solution protected from light. The noninfarcted area was red after staining, and the infarcted area was white. Each slice of the brain was placed on a piece of filter paper and photographed with a digital camera. The infarct area was measured with ImageJ software (https://imagej.nih.gov/ij/). The representative images of TTC staining are shown in Figure 4B.

4.12. Determination of Behavior Score

For all animals, a researcher blinded to the experimental group performed a series of behavioral tests before MCAO and after MCAO, and the neurological scoring system was previously described.³³ In brief, 0, no symptoms of neurological damage; 1, unable to extend the contralateral forelimb fully; 2, inability to walk straight and circling toward the ipsilateral side; 3, leaning to the affected side; 4, no spontaneous locomotor activity; 5, death. Therefore, higher scores indicated more severe injuries.

4.13. Statistical Analyses

Statistical analysis was performed using Prism (GraphPad Software, La Jolla, CA). Each result is shown as the mean ± SD. The difference between the means of the control group and treatment group was measured by t-tests, and differences with a probability less than or equal to 0.05 were considered significant.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b04129.

• LC analysis method; NMR; HRMS; mass spectra; purity analysis (PDF)

Author Contributions

^∥ S.H., R.D. and G.X. contributed equally to this work.

This work was supported by the Chengdu Science and Technology Bureau (2015-HM01-00506-SF, 2018-YF05-00454-SN).

The authors declare no competing financial interest.