Chemical & Biological Engineering
Abstract
The number of poorly water-soluble compounds has increased dramatically thanks to the advancement of high-throughput compound screening methods and combinatorial chemistry. Such compounds are difficult to formulate, and tend to precipitate in vivo due to their low solubility, leading to low bioavailability. Formulation screenings are needed to overcome the issue of drug precipitation. In vitro high-throughput precipitation methods have been developed to evaluate drug precipitation of a large pool of formulations and identify precipitation inhibitors using a limited amount of materials within a short turn-around time. This article provides a brief review of such precipitation screening methods for oral formulations.
Introduction
Approximately 40% or more of compounds identified through high-throughput compound screening are poorly water soluble [1]. Such compounds are difficult to formulate. In particular, they often tend to precipitate in vivo due to a sudden pH change from stomach to intestine, formulation dilution by body fluids, or digestion of solubilizing excipients in formulations. Such precipitation usually leads to a low oral bioavailability with a delayed response or reduced efficacy [2]. As a result, drug precipitation in vivo poses a great challenge for the development of oral formulations and dosage form development. It is crucial to assess drug precipitation in vivo during formulation screening and product development for poorly water-soluble compounds [3]. In vivo studies are naturally the most direct methods for evaluating drug precipitation of the formulations in body. However, in vivo drug precipitation are often expensive and time-consuming; they cannot be used as routine practice for formulation screening because a large number of formulations have to be screened in early drug development stage for lead formulations within a short time window. Furthermore, assessment and quantification of in vivo drug precipitation in the gastrointestinal tract are technically challenging for oral formulations and dosage forms. Therefore in vitro precipitation assays are often used for oral formulations screening.
United States Pharmacopeia (USP) dissolution methods are used for evaluation of dissolution/precipitation of oral formulations and dosage forms. They are important in vitro tests for oral product development that are required by the U. S. Food and Drug Administration (FDA) [4]. In addition, modified USP dissolution methods with multi-compartments have been reported [5-7]. They are designed to more closely mimic different regions in the gastrointestinal tract and evaluate drug precipitation in these regions. These compartments simulate stomach conditions, intestinal conditions, and absorption, respectively; therefore the effect of pH changes and absorption on drug precipitation in vivo can be assessed. These methods are easy to practice and have reasonable correlation with in vivo performance [6, 7]. However, they are low throughput methods, and require relatively large quantities of materials that would be limited in discovery and early development. There is a great need for in vitro methods that are capable of rapid drug precipitation screening for a large library of candidates.
Recently, in vitro high-throughput methods have been developed for rapid screening of drug precipitation to select the optimal formulation and rationalize the delivery system design. In addition, new analytical tools have been incorporated into precipitation methods for better monitoring and understanding of drug precipitation process. Furthermore, these methods have been used to identify novel precipitation inhibitors which are able to prevent or retard drug precipitation for improved absorption. In this short article, we focus on the development and application of the recent high-throughput drug precipitation methods for oral formulations.
Powder Methods
The powder methods are usually considered as scaled-down shake flask methods but with automation and parallel processing. Usually 96- or 384-well plates, and automatic powder dispenser and liquid handling systems are utilized. Typically, compounds are introduced to each well as solid using a solid dispenser. Then a liquid-handling system is used to dispense various combinations of excipients to each well of the plates based on formulation screening design. The plates are incubated at defined temperatures for different time periods. The solubility or concentration of compounds in the tested excipients and the combinations is determined and/or rank-ordered by optical imaging techniques or various spectroscopic methods.
High-throughput drug precipitation assays using powder methods have been developed [8]. For example, several three high-throughput screening platforms for intravenous and oral formulation screening have been reported. These platforms utilize 96- or 384-well plates with liquid volumes of as low as 5 μL per well for rapid screening. Screening of ~2,500 and 4,000-5,000 formulations per platform per day can be achieved [9]. They have been used to evaluate drug precipitation or solubilization of oral formulations to improve oral bioavailability of poorly water-soluble compounds such as celecoxib [10] and fenofibrate [11]. Powder methods have the advantages of avoiding use of organic solvents for compounds deposition, and preserving the original crystal forms of the tested compounds. They usually work the best with low viscosity excipients screening. It is challenging to apply powder methods to screen highly viscous, semi-solid and solid excipients because of the technical difficulty in dispensing and mixing such materials using common liquid/solid-handling systems. For high viscous excipients, a heating and special dispensing system may be needed for better processing of samples, which making the methods complex and costly.
Solvent Methods
To avoid the difficulty in dispensing, heating, and mixing viscous excipients, high-throughput solvent methods have been reported to evaluate drug precipitation kinetics [12-16]. Typically, compounds and a group of chosen excipients (or combinations of excipients) are first dissolved separately in selected solvents such as n-proponol [13], acetone and ethanol [16], and methanol [14]. The solutions are then dispensed into 96-well plates and mixed. After removal of solvent by evaporation, the selected biorelevant media, such as SIF (simulated intestinal fluid), FaSSIF (fasted state simulated small intestinal fluid), FeSSIF (fed state simulated intestinal fluid), or human intestinal fluid, is added to each well of the plates. The plates are incubated at room temperature for drug precipitation. At pre-set time according to the study protocol, the formulations in the plates are filtered through a 96-well plate filter under the vacuum, and drug kinetic precipitation profiles can be obtained by analyzing drug concentration in the filtrate using UV or HPLC analysis.
Among solvent methods, microscreening precipitation assay has been reported for assessing drug precipitation during formulation screening and development [12, 13]. Figure 1 illustrates the process flow of the microscreening precipitation assay. Using microscreening precipitation assay, 96 formulations with duplicate measurement in 96-well plate formats can be screened within 1-2 days per person using HPLC analysis, compared to10-30 formulations per week by a bench-top method. This assay has been used to evaluate drug precipitation of a variety of solubility-enhancing oral formulations [17]. In one study, 38 excipients were tested on a 96-well plate and ranked according to their solubilization capacity for the tested compound [12]. The lead formulation indentified by the microscreening assay was tested in pharmacokinetic (PK) studies. The rat PK study results demonstrated a significantly improved bioavailability of the lead formulation [12]. In addition, the in vitro/in vivo correlation of microscreening assay has been investigated. In one study, three formulations showing distinct in vitro precipitation kinetics (fast, slow, and no precipitation) were identified for a new molecular entity [13]. The in vitro precipitation profiles measured by the microscreening assay correlated to those determined by the USP method and canine PK studies well. The PK study showed that the fast-precipitation formulation had the lowest bioavailability among the three formulations, consistent with the screening results by the microscreening assay. In another study using solvent evaporation method, the solubility of 20 model drugs consisted of BCS I, II, III and IV types were analyzed in water and aqueous solutions (pH 1.2 and 6.8) in 96-well plates by the solvent evaporation method and shake-flask method [14]. As shown in Figure 2, the solvent method has been found to correlate well with the shake-flake method well.
Solvent methods have been also used to assess drug precipitation of self-microemulsifying formulations [15, 18]. In one study, selfmicroemulsifying formulations were created by the combination of 8 solubilizing surfactants and 8 hydrophobic oils. Using the microscrening precipitation assay, the kinetic precipitation profiles of two NMEs (aqueous solubility ≤1 μg/mL at pH 7) were assessed at four drug-loading levels (25, 50, 75 and 100 mg/g) and three incubation times (0.5, 2 and 24 h) [15]. The assay allowed rapid identification of the lead self-microemulsifying formulations and loading levels for low-solubility compounds. In another study, the self-microemulsifying formulations from the combination of 11 hydrophilic surfactants (HS) and 10 lipophilic surfactants (LS) were evaluated for optimized formulations without phase separation and drug precipitation [18]. Surfactants and the tested compound (Nilvadipine) were first dissolved in ethanol and oil (Sefsol-218) and the solution was then dispensed into 96 well plates by a robotic liquid dispenser. Drug precipitation, indicated by turbidity was assessed at each ratio of the selected HS/LS. Five lead formulations without drug precipitation were identified from a total of 2,455 formulations. The assay can screen precipitation profile of 400 formulations per person per day.
In addition, solvent methods have been used to screen and identify precipitation inhibitors which are able to prevent or retard drug precipitation and therefore maintain supersaturated drug concentration for an extended period of time. In one study, 9 surfactants were evaluated as precipitation inhibitors at concentrations below their critical micelle concentrations using microscreening precipitation assay [19]. The results showed that low concentrations of Pluronic F127 and F108 inhibited precipitation of the tested poorly water-soluble compound. Combination of Pluronics and Vitamin E TPGS resulted in a synergistic effect on drug precipitation. In another study, microscreening precipitation assay results showed that Pluronic F127 was a potent precipitation inhibitor for the labrasol formulations of two tested compounds [20].
In addition to solvent evaporation methods, a solvent shift method using 96-well plate platform has been recently reported for screening of precipitation inhibitors [21]. The process flow is illustrated in Figure 3. To start with, a 96-well plate is filled with FaSSIF containing different anti-precipitants. Then a small aliquot of the DMSO solution containing high concentration of the model drug (Itraconazole) is added to each well. After certain shaking incubation period, samples from the plates are filtered and drug concentration in the filtrate was measured by HPLC/UV. Using a solvent shift method, a precipitation inhibitor (HPMC-AS: hydroxypropyl methylcellulose-acetate succinate) was identified for Itraconazole (Figure 4), and the result was consistent with the solvent evaporation method [21]. However solvent shift method has a rapid turnaround time; only 2 hours are needed to identify the lead formulation using solvent shift method compared with 4 hours required in solvent evaporation method. The study suggests that the solvent shift method is suitable to find a desirable antiprecipitant for poorly soluble compounds.
Compared with powder methods, solvent methods avoid of difficulty in dispensing and mixing of highly viscous or solid excipients. Various biorelevant media with different pH, composition, and dilution ratios can be rapidly screened with rational design of experiments. However, solvent methods require compounds be solubilized and stable in the same volatile solvents as those for excipients during the screening process. Sometimes, it is challenging to find appropriate solvents for the tested compounds and excipients. Also drug crystalline forms may be changed after solvent evaporation in solvent evaporation methods. The compounds may be supersaturated in the excipients or exist in different polymorphic forms, leading to potential concern regarding long-term stability. For these reasons, the solvent methods are most suitable for assessing drug precipitation to identifying solubility-enhancing formulations of poorly water soluble compounds for preclinical assessments where long-term stability is not most relevant. The lead formulations identified by solvent methods can be used as starting points for the development of clinical formulation and need to be tested for long term stability.
Analytical Tools in In Vitro Drug Precipitation Methods
In addition to using conventional UV readers and HPLC methods, new analytical tools have been incorporated into in vitro precipitation methods for better understanding of drug precipitation process. For example, Arnold et al. utilized online dynamic image analysis and inline Raman spectroscopy to monitor the precipitation of dipyridamole over time [22]. A complex structure of the precipitated dipyridamole particles was revealed by the dynamic image analysis, and was described as star-like crystals or aggregates of elongated primary particles. Raman spectroscopy also enables one to measure the onset of precipitation, and determines the amount of drug precipitated as well. The precipitation data from Raman method was in good agreement with the results from HPLC analysis [22]. Compared with HPLC methods, Raman spectroscopy allows monitoring precipitation over time and provides real-time data on the precipitated drug fraction. Particle analysis tools such as dynamic light scattering have been used for analysis of precipitation and particle growth process. In a recent study, a nanoparticle tracking analysis (NTA) was utilized to demonstrate formation and growth of precipitate particles of a poorly soluble drug compound (tolnaftate, prepared with DMSO) in aqueous solution [23]. The process of precipitation can be captured by video and the information is helpful for understanding how drug precipitation occurs.
Conclusions
Drug precipitation in vivo presents a major challenge to the development of oral formulation and products for poorly water soluble drugs. in vitro high-throughput precipitation methods, such as powder and solvent methods have been used to assess drug precipitation for rapid identification of the suitable formulations for such compounds using a limited amount of materials and to accelerate drug discovery and early development. Development of new analytical tools to characterize drug precipitation will facilitate further understanding of drug precipitation mechanisms that can lead to better formulations screening.
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Author Biographies
Weiguo Dai, Ph.D., is currently a Fellow in Drug Product Development at Johnson and Johnson Pharmaceutical Research and Development. He is also an Adjunct Professor in Thomas J. Long School of Pharmacy and Health Sciences at University of the Pacific in California. Dr. Dai earned his Ph.D. degree in Chemical Engineering from The Johns Hopkins University and has over 15 years of drug product development experience in biotech/pharmaceutical companies. Dr. Dai has authored or co-authored over 40 peer-reviewed original journal publications and currently serves on the Editorial Advisory Board for a number of pharmaceutical journals. His research interests include novel drug delivery technologies and combination drug product development for both biologics and small molecules using conventional and nonconventional techniques and strategies.
Sen Xu, Ph.D., has authored more than 10 peer-reviewed scientific articles and patent applications. He earned his Ph.D. degree in Chemical Engineering from Drexel University (Philadelphia, PA) and is now with Bristol-Myers Squibb.