PROTAC Enabling Formulation In Vivo: Implications of the Polymeric Carrier Eudragit E PO

Abstract

Proteolysis targeting chimeras (PROTACs) are heterobifunctional degraders with a unique mode of action that permits access to “undruggable” targets. These molecules pose challenges in terms of solubility and bioavailability due to their physicochemical properties. So far, very little information is available on the potential of enabling formulations of PROTACs in pharmacokinetic studies. In a previous work of our group, amorphous spray-dried formulations (SDDs) of the model PROTAC MS4078 were developed. However, the potential of the amorphous solid dispersions of the PROTAC MS4078 has not yet been examined in vivo. The present study reports on the evaluation of an SDD containing MS4078 and E PO in comparison to a solution vehicle in a pharmacokinetic study in mice. Unexpectedly, very little exposure was found for both formulations. For a deeper investigation of their supersaturation and precipitation performance, the two formulations were tested in a two-stage precipitation assay with media that mimic physiological conditions in mice, which includes various bile salt and phospholipid concentrations. In this assay, the solution vehicle turned out to be a potent solubility enhancer. For the SDD, however, very different and complex dissolution profiles were obtained. The concentration at each time point was dependent on the bile salt and phospholipid concentration. Further in vitro tests revealed that E PO, the bile salt, and phospholipid interacted and induced a phase separation. This affected the solubilization and stabilization of the model PROTAC and may explain the differences regarding its in vitro performance. These findings are important to consider when designing future studies including E PO as polymeric carrier and species with high bile salt and phospholipid concentrations.

Introduction

Targeted protein degraders, also known as PROTACs, a trademark coined by the US company Arvinas, describe a group of molecules that are designed to bind to an E3 ligase, a component of the proteasome-ubiquitin system, and, simultaneously, to a protein of interest. Due to the induced proximity between the ligase and the protein, a transfer of ubiquitin onto the protein of interest is enabled, which marks it for degradation. (1,2) Because of the two functional parts of a PROTAC – each about the size of a small molecule on its own – the physicochemical properties in most cases pose a challenge in terms of solubility, lipophilicity, permeability, and consequently, oral bioavailability. (3,4) Thus, enabling formulations are likely needed for the development of oral formulations for these degraders. Despite this need, there is very little information available on formulation attempts and pharmacokinetic (PK) studies so far. In particular, there is hardly any data on pharmacokinetic studies including amorphous solid dispersions of PROTACs. (5,6) In a previous publication by our group, ASDs of a model PROTAC, MS4078, were developed and evaluated in terms of their feasibility for this modality. (7) MS4078 (structure see Figure S1) is a Cereblon-recruiting degrader of the anaplastic lymphoma kinase developed as a treatment for non-small cell lung cancers. (8) The compound was formulated in amorphous spray-dried formulations containing Eudragit E PO (E PO) or Soluplus as polymer. In both formulations, a high supersaturation was achieved in a single-stage dissolution assay in FaSSIF (fasted state simulated intestinal fluid). Relative to the neat, amorphous API, both spray-dried formulations provided a more than 70fold supersaturation.

E PO, which was used in the study as polymeric matrix, is not only a carrier in ASD formulations, (9) but also a broadly used excipient in pharmaceutical coatings. It was developed for taste and odor masking, as well as protection of the coated material from light and moisture. (10) E PO is a copolymer, with N,N-dimethylaminoethyl methacrylate, methyl methacrylate, and butyl methacrylate derived side chains (structure see Figure S1). The solubility of the polymer is strongly pH-dependent due to the presence of amine groups. In media with pH values above 5, the polymer is not dissolved, but swellable and permeable. At pH values below 5, the amine is protonated, and the polymer will be dissolved. As an ionizable polymer, E PO is prone to interact with negatively charged molecules like active pharmaceutical ingredients (APIs) (9,11) but also bile salts, which has been investigated in several studies. It has been shown that these interactions may influence the exposure in vivo. For example, Saal et al. observed a delayed release profile and influence on pharmacokinetic descriptors (cmax, tmax, AUC) after administration of a solution vehicle containing un-ionized compounds and E PO as a solubility enhancer. (12) The authors hypothesized that the pH shift upon passage to the rats’ intestines led to precipitation of both the drug and the polymer. In a different study, Schlauersbach et al. investigated the combination of bile salt interacting drugs with several polymers, including E PO. (13) The structure of FaSSIF micelles changed in the presence of the polymer due to interactions that interfered with the interplay of bile salts and the compounds. They hypothesized that the interaction might have an impact on the solubilization of bile salt interacting drugs. These publications indicate that EPO-bile salt interactions are of high relevance in vitro and in vivo.

Bile salts and phospholipids emulsify hydrophobic and lipophilic substances like pharmaceutical compounds in mixed micelles in the intestine and are crucial for their uptake. (14,15) Therefore, their implementation in biorelevant media is important for a more accurate representation of the behavior of drugs and formulations in the gastrointestinal tract. In pursuit of the best equivalent to human intestinal fluid (HIF) for in vitro assays, FaSSIF-V1, a mixture of sodium taurocholate (TC) and phosphatidylcholine (PC) (structures see Figure S1) was developed in 1998. (16) These components form colloidal structures (micelles, uni- and multilamellar vesicles of different morphologies) as equivalent to the mixed micelles in the human intestine. (17) Since then, further types have been developed, where the composition was adapted to the latest data about HIF (FaSSIF-V2, FaSSIF-V3), whereas FaSSIF-V1 remained the most popular and accepted version. Besides, adapted media for different animal species like rats have been developed (18) and an improved in vivo-in vitro-correlation (IVIVC) has been demonstrated. (19,20) Nevertheless, mice have not been characterized to the same extent, which is a huge gap since an increasing number of early PK studies are conducted in this species. (21)

The accurate representation of pH conditions in the intestine is another important factor in biorelevant dissolution and precipitation assays. There are numerous examples of pharmaceutical APIs that are weakly basic drugs like ketoconazole, ritonavir, dipyridamole, and the model PROTAC MS4078. These molecules are prone to precipitation because of pH shifts during the gastrointestinal passage. (22) Therefore, biorelevant assays that comprise two stages, one with a low pH, simulating the stomach, and a second one with a higher pH, the “intestine”, are especially valuable.
The present study reports on the evaluation of a first PK study in mice of the amorphous spray-dried dispersion (SDD) containing MS4078 and E PO (7) and the comparison to a solution vehicle (5% DMSO, 20% Kolliphor HS15 in water). For a more detailed investigation of the formulations, they were additionally tested in a two-stage precipitation assay simulating the conditions in mice. FaSSIF was prepared using a buffer system with a pH of 5 and for a more comprehensive picture, a broad range of taurocholate/phosphatidylcholine (TC/PC) concentrations was employed. In the second part, the implications of E PO as a polymeric carrier were evaluated to gain more information on the dissolution, stabilization, and precipitation of MS4078 from SDD formulations. The findings were finally compared to the in vivo results.

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Materials

The model PROTAC MS4078 was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Eudragit E PO (E PO) was provided by Evonik (Darmstadt, Germany), 3F Powder (containing bile salts (sodium taurocholate (TC)) and lecithin (phosphatidylcholine (PC)) for the preparation of FaSSIF-V1 was purchased from Biorelevant (London, UK) and Glafenine was obtained from Merck (Darmstadt, Germany). Hypergrade acetonitrile (ACN) and methanol (MeOH), analytical grade trifluoroacetic acid (TFA), dichloromethane (DCM), dimethyl sulfoxide (DMSO), Polysorbate 20 (Tween 20), sodium hydroxide (NaOH), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH2PO4), 1 N hydrochloric acid (HCl), 1 N sodium hydroxide solution (NaOH), deuterium oxide (D2O), and citric acid monohydrate were produced by Merck (Darmstadt, Germany). Methocel K4M was obtained from Colorcon (Kent, UK), Kolliphor HS 15 was provided by BASF (Ludwigshafen, Germany). All aqueous solutions were prepared with purified water (Milli-Q, Merck, Darmstadt, Germany).

PROTAC Enabling Formulation In Vivo: Implications of the Polymeric Carrier Eudragit E PO, Nicole Hofmann, Florian Johann, Katharina Krollik, Andreas Marx, Heide Marika Duevel, Marc Lecomte, Meike Harms, and Karsten Mäder
Molecular Pharmaceutics Article ASAP, DOI: 10.1021/acs.molpharmaceut.5c00303

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