Protective Effects of Quercetin against Pyrazinamide Induced

Jun 8, 2018 - Inf. Model. .... (1,3) The above two applications in the pharmacy area have been widely .... give rise to a two-dimensional grid paralle...
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Protective Effects of Quercetin against Pyrazinamide Induced Hepatotoxicity via a Cocrystallization Strategy of Complementary Advantages Fang Liu,† Ling-Yang Wang,† Yan-Tuan Li,*,†,‡ Zhi-Yong Wu,† and Cui-Wei Yan*,§ †

School of Medicine and Pharmacy and §College of Marine Life Science, Ocean University of China, 266003, Qingdao, PR China Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, 266003, Qingdao, PR China

‡

S Supporting Information *

ABSTRACT: Reducing effectively toxic side effects is a challenging subject in drug development. The reported herein cocrystallization of pyrazinamide with quercetin has complementary advantages, enhancing the in vitro/vivo performance of quercetin and taming the pharmacokinetic synergy of quercetin and pyrazinamide, almost removing pyrazinamide induced hepatotoxicity. The findings stimulate the application of active pharmaceutical ingredient−nutraceutical cocrystallization to solve the toxicity issue of drugs.

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provides medical or health benefits, including the prevention and/or treatment of a disease”.7 Therefore, their medicinal properties and potential combination with APIs should be taken seriously on cocrystallizing. However, few API− nutraceutical cocrystal designs take these into account. On the other hand, compared with the extensive research for improvement of physicochemical property and enhancement of drug therapy through the cocrystallization technique,8−11 relatively limited attention has been devoted to dealing with the toxic side effects of APIs. To the best of our knowledge, the cocrystals of API with flavonoid nutraceuticals have not been reported with the said character. With these facts in mind, in order to gain some new insights into the application of nutraceuticals in the pharmaceutical field, and furthermore to open new avenues of flavonoid nutraceuticals against drugs toxic side effects through the cocrystallization technique, recently, we have been engaged in reducing pyrazinamide (PZA) induced hepatotoxicity via cocrystallizing PZA with flavonoid nutraceuticals. It is wellknown that PZA is a first-line antitubercular drug recommended by the World Health Organization.12 But the significant liver toxicity originating from peroxidation of reactive oxygen species (ROS) impedes its clinical success.13 In particular, the treatment of tuberculosis usually requires large dose and long-term use of PZA, which brings great pain to

ocrystallization of active pharmaceutical ingredients (APIs), as an emerging technology for development of the novel pharmaceutical entity, has quickly evolved from relative obscurity to wide application in the field of pharmaceutical science and engineering.1 One of the most outstanding characters of a cocrystal is that it can offer a route to optimize APIs without altering the chemical structures and inherent bioactivities, which makes it practical to quickly develop new drugs, and has garnered the great interest of both academia and industry. 2 To date, in the context of pharmaceutical cocrystals, most attention has centered upon (1) cocrystallizing API with a pharmaceutically acceptable cocrystal coformer (CCF), namely, an API−CCF cocrystal, thus taming physical and chemical properties of the API; (2) generating combination drugs at a molecular level through API−API cocrystallization so as to obtain a superior therapeutic effect.1,3 The above two applications in the pharmacy area have been widely recognized and defined as new polymorphs of APIs and new fixed-dose combination products by the latest edition of US-FDA regulatory guidelines, respectively.4 The corresponding cocrystal drug products such as Suglat and Entresto have also been marketed.2,5 However, it is noteworthy that, in addition to the above API−CCF and API−API, another large category of pharmaceutical cocrystal, namely, “API−nutraceutical”, has regrettably not received due attention.1 So far, many studies of API−nutraceutical cocrystals focus on developing new polymorphs of APIs using the nutraceuticals as ordinary CCFs while ignoring their medicinal values. 6 In fact, nutraceuticals are accepted as “a food (or part of a food) that © XXXX American Chemical Society

Received: April 17, 2018 Revised: May 28, 2018

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DOI: 10.1021/acs.cgd.8b00576 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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patients.14 For these reasons, the hepatotoxic effects of PZA constitute a global concern, which stimulates many researchers to explore various treatment solutions.13 Unfortunately, the medical treatments for it are often difficult to manage and have limited efficacy.15 Therefore, there has been considerable interest in the role of nutraceuticals, since they may decrease the risk of toxicity of some drugs.16 Among these nutraceuticals, quercetin (QUE) as one of the most famous flavonoid antioxidants has a long history of traditional medicinal use in many countries.17 The polyphenol groups on its flavone backbone can reduce the damaging effects of oxidation caused by the ROS.18 However, there is no report about the protective effects of QUE against PZA induced hepatotoxicity, which may be mainly because of the QUE’s extremely poor aqueous solubility, hence leading to insignificant absorption and low bioavailability.19 Moreover, the potentially incompatible pharmacokinetic properties of PZA and QUE caused by the huge difference of the solubility may also further reduce the protective effect of QUE. As we all known, the protective effect of antioxidants mainly stems from reducing the oxidant in time before the injury occurs, rather than repairing the damage caused by the oxidant.20 It means that the conflict of pharmacokinetics between the two compounds, no matter the delayed absorption or premature elimination of QUE, may result in irreversible peroxidation damage induced by PZA. Thus, it is necessary for the development of new methods to increase the solubility of QUE and to tame the pharmacokinetic synergy of QUE and PZA, thereby optimizing the hepatoprotective effects of QUE. In view of these facts, we developed a cocrystallization strategy of complementary advantages to realize combination PZA and QUE at a molecular level.9,21 The strategy takes advantage of PZA’s high water solubility, to drive the increase in the solubility of QUE, thereby enhancing the QUE’s bioavailability and antioxidation effect. In turn, the optimized hepatoprotective effects of QUE, as the result of antioxidation effect enhancement, feeds back to PZA to decrease its hepatotoxicity. In addition, it is the combination of the two compounds at the molecular level after formation of cocrystal that heightens their pharmacokinetic synergy, and thus polishing the hepatoprotective effect. Along this route, an intriguing PZA−QUE cocrystal is reported herein. The PZA−QUE cocrystal described here was obtained from solvent evaporative crystallization experiments (see Supporting Information for details). Structural analysis revealed the cocrystal crystallized in a centrosymmetric P21/n space group with the asymmetric unit consisting of one molecule of QUE, one PZA, and one water molecule (Table S1 and Figure S1). As presented in Scheme 1b, two PZA molecules link each other via robust amide−amide supramolecular synthon (O8···O3(H3A), 2.926(5) Å) to form a hydrogen bonding homodimer. Moreover, the two adjacent QUE molecules are joined through O−H···O hydrogen-bonding (O4···O7(H7), 2.746(4) Å) contributing a building block. Then, two building blocks and two water molecules are arranged alternately linked by hydrogen bonds (O7···O9(H9), 2.822(3) and O5···O9(H9A), 2.208(3) Å), to form a ring structure containing six molecules (R66(51)). Furthermore, the said PZA dimer, as guest molecules, is included in the six-molecule ring, and connected to the ring through O−H···N hydrogen-bonding (N2··· O6(H6A), 2.731(3) Å). Consequentially, all these hydrogenbonding motifs give rise to a two-dimensional grid parallel to (0 0 1) crystal plane (Scheme 1b), which is additionally extended

Scheme 1. (a) Molecular Structures of QUE and PZA. (b) Crystal Structure of PZA−QUE Cocrystal

into three-dimensional architecture throughout the crystal structure via relatively weak interactions. The dissolution rate is an important physicochemical parameter for pharmaceutical solids, which has much to do with the absorption and bioavailability of orally administered drugs.22 The intrinsic dissolution rate (IDR) measurements of the cocrystal were performed in pH buffers 7.4 with pure QUE and PZA as controls by the rotating disk IDR method at 37 °C. The dissolution profiles with the calculated IDR values are given in Figure 1. It can be seen from this figure that the pure QUE shows extremely poor aqueous solubility. In fact, the concentration of QUE was even below our lower limit of

Figure 1. Release (amount versus time) of the cocrystal in pH 7.4 buffer at 37 °C in comparison to PZA and QUE from a pellet with a surface of 0.5 cm2, and calculated IDR in mg·min−1·cm−2. B

DOI: 10.1021/acs.cgd.8b00576 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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QUE in the cocrystal can also be demonstrated by the PK parameters listed in Table 1. It can be found that the CMAX and AUC of QUE in the cocrystal are 13.43-fold and 26.74-fold higher than in the physical mixture, respectively. It means that the bioavailability of QUE in the cocrystal has been significantly improved. Additionally, after the formation of cocrystal, the TMAX of QUE was advanced from 0.70 to 0.50 h, and the T1/2 was delayed from 0.85 to 1.64 h, which illustrates that the cocrystallization accelerates the absorption and meanwhile slows the elimination of QUE. In other words, the increase in the solubility of QUE through cocrystallization with PZA optimized the QUE’s PK effectively, implying a promising advantage of a hepatoprotective effect. Interestingly, in the case of PZA (Figure 2b), although the absorption rate and the peak of plasma concentration of PZA decreased after the formation of cocrystal, the FREL still maintained 0.98 (Table 1). It proved that the bioavailability of PZA did not significantly reduce with the decrease in its dissolution rate. Moreover, it is important to note that, as shown in Table 1, the elimination time of PZA and QUE was almost consistent in the cocrystal, and the TMAX of QUE was earlier than that of PZA, revealing the protective effect of QUE throughout the whole pharmacokinetic processes of PZA, which reduces the oxidant in time before the PZA induced injury occurred. As mentioned above, on the basis of the cocrystallization strategy of complementary advantages, the cocrystallization of PZA with QUE can realize the combination of the two compounds at a molecular level, displaying synergistic and optimized pharmacokinetic properties compared with the corresponding physical mixture. The above positive pharmacokinetic results for the cocrystal motivated us to further investigate the hepatoprotective effects of QUE after the cocrystal formation with PZA. For that, 24 rats were allocated into 4 groups and received orally vegetable oil (untreated controls), pure PZA, cocrystal as well as the physical mixture of PZA and QUE (equimolar ratio), respectively. The blood samples were obtained after 7 days for evaluation of total antioxidant status (TAS),25 and the activities of liver enzymes aspartate aminotransferase (AST) and alanine amino-transferase (ALT). The mean serum levels of TAS, AST, and ALT for the four groups are shown in Figure 3. As illustrated in Figure 3a, the TAS value was significantly decreased due to the treatment with pure PZA (without QUE) compared with untreated controls, indicating that taking PZA could seriously break the balance between prooxidants and antioxidants, hence injuring the liver.26 While the coadministration of QUE with PZA as the form of physical mixing produces some increase in TAS value owing to the antioxidant capacity of QUE, there is still an obvious gap compared with that reported in the untreated control, which may be mainly because of the confirmed poor bioavailability of QUE. It is noteworthy that treatment with the cocrystal, by contrast, can highly increase

detection at the initial three points of time. In contrast, the PZA exhibits a remarkable dissolution advantage with much higher dissolution rates than QUE. The IDR value of the cocrystal is located in between pure QUE and PZA, and is closer to the value of pure PZA. Obviously, the advantage of PZA’s high water solubility drives the increase of QUE’s solubility after the formation of cocrystal. As a result, the IDR value of cocrystal improves 33-fold over pure QUE (Figure 1). From the structural point of view, the enhanced solubility of the cocrystal could be explained by the fact that, on being exposed to an aqueous environment, the more soluble PZA and water molecules in the cocrystal leach into the solution from the crystal lattice, thereby leaving behind the QUE framework which may be floating in the solution medium with loose selfaggregation. This amorphous-like state of the QUE molecules quickly dissipates in the solvent, resulting in the observed high dissolution rate of QUE.23 In conclusion, the advantage of PZA’s high water solubility improves the dissolution properties of the QUE in the cocrystal significantly, which lays the foundation for enhancing the bioavailability of QUE and further reducing the toxicity of PZA. After satisfactory in vitro release studies, the pharmacokinetics (PK) properties were evaluated to examine the changes of in vivo performance of the cocrystal in comparison with the physical mixture of PZA and QUE (equimolar ratio) by oral administration of the cocrystal/mixture to Sprague−Dawley rats.19 The serum concentration profiles are given in Figure 2,

Figure 2. Pharmacokinetic profiles of (a) quercetin and (b) pyrazinamide (mean plasma concentration ± SD versus time).

and the PK parameters determined by PKSolver software,24 are illustrated in Table 1. As viewed from Figure 2a, the QUE in the physical mixture was absorbed very poorly, which is easily explained by the extremely poor solubility of QUE. Instead, in the case of the cocrystal, the QUE exhibited favorable pharmacokinetic profiles, which is consistent with the changes in the solubility. The optimized pharmacokinetic properties of

Table 1. Pharmacokinetic Parameters Determined by PK Solutionsa TMAX (h) CMAX (μg·mL−1) AUC0‑t (μg·h·mL−1) AUC0‑inf (μg·h·mL−1) FREL T1/2 (h) a

QUE in cocrystal

QUE in mixture

PZA in cocrystal

PZA in mixture

0.50 (0.18) 8.73 (1.20) 15.51 (4.21) 17.07 (5.71) 26.74 1.64 (0.40)

0.70 (0.11) 0.65 (0.27) 0.58 (0.27) 0.66 (0.28) −/− 0.85 (0.24)

1.60 (0.55) 122.82 (28.65) 364.47 (30.11) 391.73 (33.91) 0.98 1.66 (0.45)

0.95 (0.11) 155.04 (10.02) 372.42 (18.29) 380.07 (18.52) −/− 1.24 (0.37)

SD for five readings is given in parentheses. C

DOI: 10.1021/acs.cgd.8b00576 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Accession Codes

CCDC 1834049 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*(Y.-T. Li) E-mail: [email protected]. *(C.-W. Yan) E-mail: [email protected]. ORCID

Yan-Tuan Li: 0000-0002-5225-725X Notes

The authors declare no competing financial interest.

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Figure 3. Protective effects of quercetin against pyrazine-amide in cocrystal and mixture detected by serum (a) TAS and (b) liver enzymes level in rats (n = 6).

ACKNOWLEDGMENTS We thank the Key Research and Development Plan of Shandong Province (No. 2018GSF118174) and the NSFCShandong Joint Fund for Marine Science Research Centers (No. U1606403).

the TAS value, and the achieved value was comparable to that reported in the control. These results suggested that the cocrystal formation markedly enhances the QUE’s antioxidant ability, optimizes hepatoprotective effects, and protects the liver from damage, which is further proved by the changes in serum levels of liver enzymes. As indicated in Figure 3b, treatment of rats with PZA produces a significant increase in serum AST and ALT activities as a result of PZA induced liver damage. While in the case of the cocrystal, the elevations in serum levels of AST and ALT activities are significantly attenuated and realizing full protection with serum levels of liver enzymes (both AST and ALT) comparable to those of untreated controls. The protection of cocrystal detected both by diagnostic indicators of liver injury (AST and ALT levels) and by TAS can prove the fact that the cocrystallization of QUE with PZA through the strategy of complementary advantages almost removed PZA induced hepatotoxicity, suggesting a promising advantage of clinical effects. It is one of the eternal themes of drug development to effectively reduce APIs’ toxic side effects. In this report, the antituberculosis drug PZA was cocrystallized with nutraceutical QUE via the strategy of complementary advantages, so as to utilize the protective effects of QUE against PZA induced serious hepatotoxicity. The API−nutraceutical cocrystal was examined for aqueous dissolution, in vivo PK properties, and toxicity to predict its enabled clinical efficacy. The results showed that, as the strategy predesigned, the PZA−QUE cocrystal enhanced the in vitro/vivo properties of QUE and improved the pharmacokinetic synergy of QUE and PZA, hence almost removing the hepatotoxicity of PZA. Thus, the present studies provide a new thinking for the application of insoluble nutraceuticals in the pharmaceutical field, and open new avenues of flavonoid nutraceuticals against drugs’ toxic side effects through the cocrystallization technique.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.8b00576. Experimental, spectroscopic, and crystallographic details (PDF) D

DOI: 10.1021/acs.cgd.8b00576 Cryst. Growth Des. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.cgd.8b00576 Cryst. Growth Des. XXXX, XXX, XXX−XXX