A carrier free delivery system of a monoacylglycerol lipase hydrophobic inhibitor
Graphical abstract
Introduction
Drug design and combinatorial chemistry have been exploited to develop new drugs, which are generally characterized by high lipophilicity that assist drugs to transfer across membranes (Verma et al., 2009) and therefore low solubility in water (Kalepu and Nekkanti, 2015). Indeed 40–70% of new molecules developed in the context of drug discovery programs are not enough soluble in aqueous media, thus compromising the bioavailability of the drug (Gupta et al., 2013). Therefore, in order to became commercially available (in general almost 10–17 years after its first synthesis (Barbosa et al., 2019)) a poorly soluble drug needs to be formulated through drug delivery technologies.
Many drug discovery programs are dedicated to cancer, which is the second major disease in Europe that kills millions of people around the globe every year. Indeed, despite several therapeutic strategies have been employed to cure cancer, there is still no accurate ways to eradicate it by roots (Falzone et al., 2018, Hanahan and Weinberg, 2011) and chemotherapy, which is considered one of the major treatment tools, suffers from several issues and even failures (Adeel et al., 2020, Liao et al., 2020, Mirza and Siddiqui, 2014). To overcome chemotherapy issues, current approaches are focused on the discovery of drugs that target specific receptors or proteins mostly upregulated in tumor cells. This strategy takes part of a wider concept termed personalized therapy in which each patient is treated with specific targeted drugs based on the genotype and phenotype. Several research groups are exploring many genes, receptors, and enzymes that are commonly involved in many cancer types and could be a therapeutic target (Fabbro et al., 2002, Falzone et al., 2018, Fleck and Bach, 2012). Among the enzymes, monoacylglycerol lipase (MAGL) is a member of the serine hydrolase superfamily, which primarily degrades the endocannabinoid 2-arachidonoylglycerol (2-AG) to arachidonic acid and glycerol in the brain, thus interrupting its signaling on cannabinoid receptors CB1 and CB2. MAGL is also involved in the hydrolysis of monoacylglycerols in peripheral tissues (Long et al., 2009b). For this reason, in cancer cells MAGL was reported to activate oncogenic signaling related to the intracellular pool of free fatty acids (Nomura et al., 2010) and it proved to be involved in sustaining the growth of different cancer cells (Bononi et al., 2021, Granchi et al., 2017, Granchi et al., 2016).
Several MAGL inhibitors are reported in literature, which can be classified according to the mechanism of action: a) irreversible inhibitors, binding permanently the enzyme: they are generally characterized by an high inhibition potency, but they induce some undesired effects in vivo (loss of CB1-mediated biological effects and CB1 desensitization); b) reversible inhibitors, which interact with the enzyme blocking temporarily its catalytic function, without provoking the previously mentioned negative effects. Among the irreversible MAGL inhibitor, we can mention the JZL184 (Long et al., 2009a) and the most recent ABX1431 (Cisar et al., 2018) which is currently under clinical evaluation. One of the first reversible MAGL inhibitors is a benzo[d] [1,3]dioxol-5-ylmethyl 6-phenylhexanoate derivative discovered by Hernández-Torres et al. (Hernández-Torres et al., 2014). Some of the most representative reversible MAGL inhibitors are the class of azetidine-based derivatives developed at Janssen Research & Development, L.L.C. (Zhu et al., 2020b, Zhu et al., 2020a), piperazinyl pyrrolidin-2-ones and spiro compounds developed at Takeda Pharmaceutical Co., Ltd. (Aida et al., 2018, Ikeda et al., 2021), salicylketoxime (Bononi et al., 2018) and aryl formyl piperidine derivatives (Zhi et al., 2020).
Recently, our group developed a reversible and selective MAGL inhibitor (MAGL23, Fig. 1), which inhibits the target enzyme with a potency in the nanomolar range (Ki = 39 nM) (Granchi et al., 2019). MAGL23 is a compound with suitable properties to be further developed as a drug because it is obtained by a few steps’ synthetic preparation. Moreover, its reversible binding mechanism makes it devoid of the typical side effects of irreversible MAGL inhibitors observed in in vivo studies (Schlosburg et al., 2010). Finally, it also showed selectivity for MAGL inhibition over the other main components of the endocannabinoid system, i.e., cannabinoid receptor 1 and 2, fatty acid amide hydrolase, α/β hydrolase-6 and α/β hydrolase-12. Interestingly, the ability of MAGL23 to inhibit the viability of ovarian, colon, and breast cancer cell lines was demonstrated (Granchi et al., 2019).
Although the results in terms of antiproliferative activity in cancer cells were quite good (IC50 values in the low micromolar range), the inhibitor suffered of problems related to poor solubility. Recently, nanotechnology is playing a vital role to improve important drug parameters such as solubility, biodistribution, pharmacokinetics and pharmacodynamics (Bayda et al., 2018, Kumar et al., 2016, Lepeltier et al., 2020, Palazzolo et al., 2018, Rizzolio, 2018). Among the possible approaches to increase the solubility, nanocrystallization allows to avoid a matrix such as polymeric or lipid nanoparticles. Indeed, drug nanocrystal particles have an almost 100% hydrophilic surface, avoiding the toxic effects of the matrix and with a high yield of solubilization (Liu et al., 2010, Müller et al., 2011). In nanotechnology, two different approaches can be exploited: the “Top-down” and “Bottom-up” methods. When applied to crystals, the main concept is to reduce their size from several microns to a few nanometers that are easily soluble in water. The “top-down” approach foresees the use of a high shear stress for the breakage of large crystals while in the “bottom-up” approach, the nanocrystals are generated from the crystallization of drug molecules. Furthermore, the two processes can be used one after the other in a combination process, where usually after a “bottom-up” process of crystallization, size is decreased with a “top-down” method such as ultrasonication (El Hadri et al., 2020, Fontana et al., 2018). However, these methods still have limits due to the variation in the size of nanocrystals during batch preparation and the chemical nature of the compounds (Du et al., 2015, El Hadri et al., 2020, Mahesh et al., 2014, Park et al., 2017).
Recently, Park et al. introduced a novel nanocrystallization strategy for the solubilization of poorly soluble drugs in water by a combined method. The authors used the Pluronic F-127 and cetyltrimethylammonium bromide (CTAB) as surfactants to obtain a controlled generation of nanocrystals in water and then covered them with human serum albumin (Park et al., 2017). The presence of surfactant during the crystallization allows to control the size of the obtained nanocrystals. More importantly, the bare nanocrystals of therapeutic compounds can lead to the adsorption of non-specific proteins resulting in the uptake by the mononuclear phagocyte system (MPS) (Parviz et al., 2020, Walkey et al., 2012). Despite the fact that others hydrophilic stealth surfaces could reduce the MPS uptake of nanocrystals, there would always be a possibility that after the distribution of nanocrystals in tumors, they could interfere with the retention and intended cellular interactions (Hatakeyama et al., 2013). On the contrary, albumin is a natural carrier of native ligands and other hydrophobic molecules, it is very stable, digestible and decomposable by cells, provides amino acids for cell metabolism and helps to internalize the nanocrystals in tumoral sites through the interaction with specific receptors (Daneshamouz et al., 2021, Hoogenboezem and Duvall, 2018, Li et al., 2020, Parodi et al., 2019, Stehle et al., 1997).
In the proposed study, it was utilized a combined method to obtain nanocrystals of the MAGL23 inhibitor that was highly hydrophobic. The inhibitor was solubilized with Pluronic F-127 or CTAB surfactants in the organic solvent (chloroform) and the crystals were obtained by sonication in water. We compared the effects of surfactants and the interaction of albumin with the MAGL23 inhibitor. The system was observed at each step of the formulation by using different spectroscopic techniques to ascertain the stability of the formulation. We believe that the proposed system provides an opportunity to increase the solubilization of poorly soluble hydrophobic drugs, such as MAGL23 inhibitor, and create a drug delivery system for in vivo applications.
Section snippets
Reagents
Lyophilized powder of albumin from human serum albumin (HSA) ≥ 96% (Cat n° A3782), hexadecyltrimethylammonium bromide (CTAB) ≥ 98% (Cat n° H5882), powder of Pluronic® F-127 (Cat n° P2443) suitable for cell culture and acetonitrile (Cat n° 151807) were purchased from Merck, Darmstadt, Germany. To perform Bradford protein assay, dye reagent (Cat n° 500–0006) was purchased from Bio-Rad, Hercules, CA, US; methanol (Cat n° A177150010) for LC/MS was purchased from Carlo Erba, Milan, Italy; chloroform
Nano crystallization process increases the solubility of MAGL23
MAGL23 is an organic molecule with a medium molecular weight (369.436 g/mol) which, basing on the Chemicalize.com prediction (predicted physicochemical properties are reported in Table 1), is highly hydrophobic and insoluble at physiological pH levels (Fig. 2a) at which it is mainly in its neutral form (94% at pH = 7.4) (Fig. 2b). In order to confirm the poor solubility of MAGL23 at neutral pH, a solubility test was performed on drug powder, following the method reported in section 2.2. The
Conclusions
The first nanoformulation of a potent MAGL inhibitor, MAGL23, which is a promising compound as anticancer agent, was developed. MAGL is an enzyme that is highly expressed in tumors, where it plays a fundamental role in the oncogenic lipid signaling that promotes invasion, migration, survival, and in vivo tumor growth (Nomura et al., 2010). Many MAGL inhibitors have been reported in the literature and they showed positive effects when tested in in vivo studies. However, most MAGL inhibitors act
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was financially supported by SPIN-Supporting Principal Investigators, Ca’Foscari University of Venice, Italy; by MIUR (PRIN 2017, project 2017SA5837) and by the Italian Ministry of Health – Ricerca Finalizzata 2016 - NET-2016-02363765.
References (65)
- et al.
Niclosamide repositioning for treating cancer: Challenges and nano-based drug delivery opportunities
Eur. J. Pharm. Biopharm.
(2019) - et al.
Discovery of long-chain salicylketoxime derivatives as monoacylglycerol lipase (MAGL) inhibitors
Eur. J. Med. Chem.
(2018) - et al.
Protein-bound uremic toxins (PBUTs) in chronic kidney disease (CKD) patients: Production pathway, challenges and recent advances in renal PBUTs clearance
NanoImpact
(2021) - et al.
Nanosuspensions of poorly water-soluble drugs prepared by bottom-up technologies
Int. J. Pharm.
(2015) - et al.
Nanoplastic from mechanically degraded primary and secondary microplastics for environmental assessments
NanoImpact
(2020) - et al.
Synthesis and characterization of biocompatible hydrogel using Pluronics-based block copolymers
Polymer (Guildf)
(2013) - et al.
Impact of albumin on drug delivery - new applications on the horizon
J. Control. Release.
(2012) - et al.
Protein kinases as targets for anticancer agents: from inhibitors to useful drugs
Pharmacol. Ther.
(2002) - et al.
Production of pure drug nanocrystals and nano co-crystals by confinement methods
Adv. Drug Deliv. Rev.
(2018) - et al.
Monoacylglycerol lipase (MAGL) as a promising therapeutic target
Biochem. Pharmacol.
(2018)
Hallmarks of cancer: the next generation
Cell
Harnessing albumin as a carrier for cancer therapies
Adv. Drug Deliv. Rev.
A clinical update of using albumin as a drug vehicle - A commentary
J. Control. Release.
Smart nanocarriers for cancer treatment: clinical impact and safety
NanoImpact
Targeted cancer therapy with novel high drug-loading nanocrystals
J. Pharm. Sci.
Characterization of monoacylglycerol lipase inhibition reveals differences in central and peripheral endocannabinoid metabolism
Chem. Biol.
A comparative study of top-down and bottom-up approaches for the preparation of nanosuspensions of glipizide
Powder Technol
State of the art of nanocrystals - Special features, production, nanotoxicology aspects and intracellular delivery
Eur. J. Pharm. Biopharm.
Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis
Cell
Potential of Pluronics ® P-123 and F-127 as nanocarriers of anti-Leishmania chemotherapy
Acta Trop
Pharmacological inhibition of MAGL attenuates experimental colon carcinogenesis
Pharmacol. Res.
Albumin-coated nanocrystals for carrier-free delivery of paclitaxel
J. Control. Release.
Engineering two-dimensional nanomaterials to enable structure-activity relationship studies in nanosafety research
NanoImpact
Preformed albumin corona, a protective coating for nanoparticles based drug delivery system
Biomaterials
Plasma protein (albumin) catabolism by the tumor itself - Implications for tumor metabolism and the genesis of cachexia
Crit. Rev. Oncol. Hematol.
A comparative study of top-down and bottom-up approaches for the preparation of micro/nanosuspensions
Int. J. Pharm.
Local drug delivery strategies for cancer treatment: gels, nanoparticles, polymeric films, rods, and wafers
J. Control. Release.
The discovery of diazetidinyl diamides as potent and reversible inhibitors of monoacylglycerol lipase (MAGL)
Bioorg. Med. Chem. Lett.
The discovery of azetidine-piperazine di-amides as potent, selective and reversible monoacylglycerol lipase (MAGL) inhibitors
Bioorg. Med. Chem. Lett.
Self-therapeutic nanomaterials for cancer therapy: a review
ACS Appl. Nano Mater.
Design, synthesis, and evaluation of piperazinyl pyrrolidin-2-ones as a novel series of reversible monoacylglycerol lipase inhibitors
J. Med. Chem.
New method for delivering a hydrophobic drug for photodynamic therapy using pure nanocrystal form of the drug
Mol. Pharm.
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