Interactions between dipfluzine-based complexes and cytochrome P450 enzymes: Information on salt, cocrystal, and salt cocrystal complexes

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Highlights

  • Crystalline molecular complexes has become a shortcut for new drug development.

  • Interaction between CMCs and CYP enzymes were investigated.

  • CMCs alter the effects of Dip on CYP enzymes and the degree of rCYPs.

  • Interaction forces between API and CCF alter the interaction of Dip and CYP enzymes.

  • Re-evaluating the interactions between CMCs and CYP enzymes is necessary.

Abstract

In the new drugs, greater than 90 % of active pharmaceutical ingredients (APIs) or marketed drugs have poor solubility and bioavailability, which restrict the development of pharmaceutical preparations. The use of crystalline molecular complexes (CMCs) involving API and biocompatible precursors to improve solubility has become a shortcut for new drug development. Most of the new drugs registered in CMC form are from postmarketing drugs, and the interaction between these drugs and cytochrome P-450 (CYP) enzymes is well documented. However, it is unclear whether the interactions between CMCs of postmarketing drugs and CYP enzymes should be re-evaluated. To clarify this problem, three dipfluzine (Dip)-based CMCs, including Dip-benzoic acid (BA) cocrystal, Dip-2-hydroxybenzoate (2HB) salt and Dip-4-hydroxybenzoate (4HB) salt-cocrystal, were chosen to investigate the interaction with CYP enzymes. Metabolites of Dip-based CMCs and cocktail probe drugs were measured via LC–MS/MS in the incubation reaction system comprising recombinant CYP enzymes (rCYPs) and human liver microsomes. Dip-based CMCs not only alter Dip-mediated inhibition or activation of CYP enzymes but also change the degree to which rCYPs are involved in Dip metabolism. Specifically, the inhibitory effects of Dip and Dip-HCl were increased compared with Dip-BA and Dip-2HB for CYP1A2; Dip-BA, Dip-2HB and Dip-4HB for CYP3A4; and Dip-BA for CYP2E1. The inhibitory effects of Dip and Dip-HCl were reduced compared with Dip-2HB and Dip-4HB for CYP2C19 and Dip-4HB for CYP2E1. The effects of the alterations of Dip CMCs on the interaction between Dip and CYP enzymes are not attributed to a simple superposition of Dip and the respective precursor and may be due to the presence of interaction forces between Dip and precursor molecules. These results are the first to provide preliminary experimental evidence that CMCs change the interaction between API and CYP enzymes. Moreover, these results further suggest the importance of re-evaluating interactions with CYP enzymes when CMC strategies are used to design new drugs and even for CMCs of postmarketing drugs with known metabolic characteristics.

Introduction

Polypharmacy has become a main means of clinical treatment. Activators, inhibitors or substrates of the same drug metabolizing enzymes (DME) in the polypharmacy pose a risk of drug-drug interactions (DDIs) related to DME (Li et al., 2013). When the metabolism of one drug can be changed by another drug, DDIs can change the dose-response relationship and lead to reduced efficacy, the treatment failure and/or increased toxicity. Therefore, determining whether the drug is the substrate, inhibitor or activator of DME in clinical treatment is important, especially in the case of the polypharmacy (Nagai, 2010; Huang et al., 2008). The FDA has started to explore the interactions between candidate drugs and cytochrome P-450 (CYP) enzymes to predict the DDI risk given that this information is important for new drug development, clinical drug use and legal issues.

Generally, greater than 90 % of drugs and approximately 40 % of active pharmaceutical ingredients (APIs) or postmarketing drugs display poorly water solubility (Hauss, 2007) and the bioavailability of orally administered drugs noticeably depends on solubility. Therefore, improving the solubility of APIs is one of the most challenging tasks in oral drug design. Crystal engineering offers potential methods to improve the solubility and dissolution rate of APIs, such as the formation of crystalline molecular complexes (CMCs). Pharmaceutical CMCs are a complex system formed by the combination of API molecules and biocompatible small molecular precursors through ionic bonds and/or weak hydrogen bonding forces, which yield a variety of distinct solid forms at room temperature, including salt, cocrystal and salt-cocrystal (Vishweshwar et al., 2006; He et al., 2017).

To date, it is not clear whether an API-based CMC should be defined as a new chemical entity requiring full safety and toxicological assessment or whether information from their parent physical mixture is suitable (Brittain, 2019). Fortunately, researchers are realizing the problem and voicing concerns about performing appropriate investigations before using CMCs to improve API bioavailability. Ferretti et al. reported that indomethacin-based cocrystals exhibited different values of the transepithelial electrical resistance (TEER) and abilities to permeate across NCM460 cell monolayers compared with the parent physical mixture of API and cocrystal former (CCF) (Ferretti et al., 2015). In our previous study, investigations on binding interactions between the gallic acid-glutaric acid complex and α-glucosidase indicated that the gallic acid-glutaric acid complex deeply entered the active cavity of α-glucosidase in the form of a gallic acid-glutaric acid supramolecule, thereby changing the binding energy of the guest-enzyme conformation and further increasing the enzyme activity of α-glucosidase (Xue et al., 2020). These findings suggest that the introduction of precursor molecules in CMC construction induce changes in the biological profiles of the API.

An interesting question regarding drug combinations and the risk of DDI arises: Do the intermolecular forces between the API and precursor affect the interaction between the API and DME if the intermolecular forces in the API-CMC system are maintained during transport? Unfortunately, the answers for most API-CMC researchers are negative. It is generally believed that after absorption in the body, CMCs assume the chemical form of the free API molecule (Walsh et al., 2018; Abidi et al., 2018), and the interaction mechanism between API or its CMC and DME, such as CYP enzymes, should be similar. Thus, the pharmacological behaviour of API, including the interaction between API and the CYP enzyme in vivo, should not change. However, based on our previous investigation on the binding interaction between gallic acid-glutaric acid complex and α-glucosidase (Xue et al., 2020), we hypothesize that the introduction of a precursor in the CMC may affect API metabolism by activating or inhibiting CYP activity.

Dipfluzine (Dip) is a novel diphenylpiperazine Ca2+ channel blocker that was synthesized by the Pharmaceutical School of Hebei Medical University. Compared with its analogue cinnarizine or flunarizine, Dip effectively induces selective cerebrovascular dilation, reduces thrombosis, lowers cerebral oedema, ameliorates ischaemic brain injury, and significantly repairs memory impairment caused by sodium nitrite in mice (Wang and He, 1993, 1994a, 1994b; Mei et al., 2004; Chen et al., 2005; Li et al., 2008; Zhu et al., 1996). However, Dip is a poorly water-soluble API. To improve its solubility in water, we used hydrochloric acid (HCl), benzoic acid (BA), 2-hydroxybenzoate (2HB), and 4-hydroxybenzoate (4HB) as precursors to obtain four Dip CMCs through CMC screening experiments, including one cocrystal (Dip-BA), two salts (Dip-HCl and Dip-2HB), and one salt-cocrystal (Dip-4HB). The solubility, dissolution rate and relative bioavailability of Dip were improved. For example, the oral bioavailability of Dip-BA cocrystal increased approximately 2-fold compared with Dip and without its distribution reducing in the brain (Lin et al., 2014; Du et al., 2018). However, whether the introduction of precursors, namely, BA, 2HB, and 4HB molecules, alter the activities of some CYP enzymes remains unclear.

Previous studies have demonstrated that Dip and Dip-HCl not only transformed five metabolites (M1-M5) produced by CYP enzymes in vitro and in vivo but also induced or inhibited the activities of some CYP enzymes (Liu et al., 2005; Hu et al., 2009; Guo et al., 2012; Guo et al., 2014a, 2014b). These results indicate a probable risk of DDI correlated with CYP enzyme metabolism when Dip or Dip-HCl is combined with other drugs that inhibit or induce the same CYP isoenzymes. In the present study, three Dip-based CMCs, including Dip-BA cocrystal, Dip-2HB salt and Dip-4HB salt-cocrystal, were chosen to investigate the interaction with CYP enzymes. Metabolites of Dip-based CMCs and cocktail probe drugs were measured using LC–MS/MS in the incubation reaction system comprising human recombinant CYP (hrCYP) enzymes and human liver microsomes. We focused on the change in the interaction with CYP enzymes induced by the complexation of Dip with BA, 2HB, and 4HB.

Section snippets

Chemicals

Nicotinamide adenine dinucleotide phosphate (NADPH) and Phosphate buffer solution (PBS) were purchased from Beijing Solebo Reagents Co., Ltd. Human liver microsomes and nine hrCYPs (hrCYP1A2, hrCYP2A6, hrCYP2B6, hrCYP2C8, hrCYP2C9, hrCYP2C19, hrCYP2D6, hrCYP2E1 and hrCYP3A4) were purchased from BD Company (Woburn, MA). Tolbutamide, 4-hydroxytolbutamide, dextromethorphan, dextrorphan, omeprazole, 5-hydroxyomeprazole, magnesium chloride (MgCl2), coumarin, 7-hydroxycoumarin, chlorzoxazone,

Effects of dip-based CMCs on the contributions of human recombinant CYP enzymes in Dip metabolism

Dip and its CMCs were incubated with a panel of hrCYPs, including hrCYP1A2, hrCYP2A6, hrCYP2B6, hrCYP2C8, hrCYP2C9, hrCYP2C19, hrCYP2D6, hrCYP2E1, and hrCYP3A4, respectively, at a protein concentration of 1 mg/mL in 50 mM potassium phosphate buffer (pH 7.4) to assess the contribution of each individual hrCYP enzyme in Dip metabolite (M1 and M5) formation. Results are shown in Fig. 2. Since M1 and M5 are the two first-order metabolites, their production rates are the main indexes used to detect

Conclusion

The metabolites of Dip-based CMCs and cocktail probe drugs in the incubation reaction system comprising human liver microsomes and recombinant CYP enzymes (rCYPs) were measured using LC–MS/MS techniques. Compared with Dip, the introduction of precursors not only altered the effect of Dip on CYP enzyme inhibition or activation but also altered the degree to which hrCYPs are involved in Dip metabolism. Interestingly, the effects of Dip CMCs on CYP enzyme activity were not attributed to the simple

CRediT authorship contribution statement

Huan Wang: Methodology, Validation. Shiji Li: Formal analysis, Project administration. Lili Liu: Investigation. Jing Wang: Visualization, Supervision. Yongli Wang: Data curation, Writing - original draft. Wei Guo: Software, Conceptualization, Writing - review & editing.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

This work received financial support from the Natural Science Foundation of Hebei Province in 2017 (No. 2017206261) and the Youth Fund for Scientific and Technological Research in Higher Institutions of Hebei Province in 2017 (NO. QN2017100).

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