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From Molecule to Dose Form – Accelerated Bioavailability Enhancement for Early Phase Molecules A special article collection companion to the webinar broadcast October 21st, 2015. To view the webinar, click here Sponsored by http://www.catalent.com/ Image courtesy of Catalent Pharma Solutions. © 2016 Catalent, Inc. All rights reserved Table of Contents Introduction Professor Juergen Siepmann, PhD University of Lille, College of Pharmacy pages 1-3 Strategies for formulating and delivering poorly water-soluble drugs Journal of Drug Delivery Science and Technology, Volume 30, Part B, December 2015, Pages 342-351 Marta Rodriguez-Aller, Davy Guillarme, Jean-Luc Veuthey, Robert Gurny pages 4-13 Amorphous drugs and dosage forms Journal of Drug Delivery Science and Technology, Volume 23, Issue 4, 2013, Pages 403-408 H. Grohganz, K. Löbmann, P. Priemel, K. Tarp Jensen, K. Graeser, C. Strachan, T. Rades Recent developments in oral lipid-based drug delivery Journal of Drug Delivery Science and Technology, Volume 23, Issue 4, 2013, Pages 375-382 N. Thomas, T. Rades, A. Müllertz pages 14-19 pages 21-28 Introduction J. Siepmann1,2 1 2 Univ. Lille, F-59000 Lille, France INSERM U 1008, F-59000 Lille, France *correspondence: Professor Juergen Siepmann, PhD University of Lille, College of Pharmacy INSERM U 1008 3 Rue du Prof. Laguesse 59006 Lille, France Fax: +33-3-20964942 [email protected] Nowadays, one of the major hurdles to be faced during the development of innovative drug products is the limited bioavailability of numerous drug candidates. This is in great part due to the very low aqueous solubility of these compounds. Consequently, they do not have the time to dissolve to a sufficient extent upon administration to the living organism. Importantly, only dissolved drug is able to diffuse and cross major barriers in the human body, e.g. the mucosa in the gastro intestinal tract. Thus, even if the chemical structure of the drug candidate is ideal to allow for efficient interaction with its target (and cure the patient), the compound fails in vivo, since it is not able to reach its site of action. A broad range of formulation approaches has been proposed to overcome this crucial bottleneck, aiming at increasing the dissolution rate of poorly water-soluble drugs upon administration into the living body. In order to quantify the dissolution rate of a drug particle, the famous Noyes-Whitney equation can be used [1,2]: 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 = 𝐾𝐾(𝑐𝑐𝑠𝑠 − 𝑐𝑐𝑡𝑡 ) (1) where dc/dt is the dissolution rate; K is a constant; cs denotes the solubility of the substance, and ct is the concentration of dissolved substance in the surrounding bulk fluid at time t. The basic hypothesis is that the diffusion of individualized drug molecules, ions or atoms through the liquid unstirred boundary layer surrounding the drug particle is the slowest step in the Page 1 1 series of events occurring during drug particle dissolution [3]. Thus, this step (which is illustrated in Figure 1) is rate limiting and governs the dissolution kinetics. unstirred layer cs ct Figure 1: Schematic presentation of the mass transport process, which is often dominant during the dissolution of a drug particle: The diffusion of individualized drug molecules/atoms/ions through the unstirred liquid boundary layer surrounding the drug particle (adapted from [3]). Nernst and Brunner [4,5] used Fick’s first law of diffusion to quantify this diffusional mass transport step and derived the following equation: dM S ⋅ D = ⋅ (c s − ct ) dt d (2) where dM is amount of substance which dissolves in the time interval dt; S denotes the surface area available for diffusion/dissolution; D is the diffusion coefficient of the drug within the liquid unstirred boundary layer; d is the thickness of this layer; cs and ct are the solubility of the drug in the bulk fluid and the concentration of dissolved drug in the bulk fluid at time t, respectively. Looking at the Nernst-Brunner equation (Equation 2), it becomes obvious that different strategies can be used to increase the dissolution rate of a drug. In particular, one can aim at: (i) increasing the surface area available for dissolution via a reduction of the particle size; and/or (ii) increasing the apparent solubility of the drug in the surrounding environment, e.g. Page 2 2 via transformation into a physical state with a higher energy. These first two strategies are very often applied. Eventually, the aim might also be to keep the concentration of dissolved drug in the surrounding bulk fluid low, e.g. by facilitating the subsequent drug transport away from its site of dissolution. On the other hand, the thickness of the liquid, unstirred boundary layer as well as the diffusion coefficient of the drug in this layer are generally very difficult to alter in practice in the human body. This ebook is a selection of articles published in the Journal of Drug Delivery Science and Technology, giving overviews on different types of strategies that are used to increase the dissolution rates of poorly water-soluble drugs in order to increase their bioavailability. A variety of practical examples are given and limitations of the different approaches are discussed. The article of Grohganz et al. reviews the current state of the art in the field of amorphous drug forms. The basic idea is to provide the drug in a physical form with a high energy (and, thus, higher apparent solubility). However, recrystallization during long term storage is a major concern, which needs to be addressed. The review article by Thomas et al. gives a comprehensive overview on the latest developments in oral lipid-based drug delivery systems. Briefly, in these cases highly lipophilic drugs are dissolved in lipid dosage forms, thus, avoiding the drug dissolution step in the human body (or in other words: the apparent drug solubility in the dosage form is so much increased that the entire drug dose is already dissolved). Finally, the article by Rodriguez-Aller et al. gives a comprehensive overview on the broad variety of approaches used to better formulate poorly water-soluble drugs, including many examples of drug products which are available on the market. References [1] Noyes, A.A., Whitney, W.R., 1897. The rate of solution of solid substances in their own solutions. J. Am. Chem. Soc. 19, 930-934. [2] Noyes, A.A., Whitney, W.R., 1897. Ueber die Aufloesungsgeschwindigkeit von festen Stoffen in ihren eigenen Loesungen. Z. Physikal. Chem. 23, 689-692. [3] Siepmann, J., Siepmann, F., 2013. Mathematical modeling of drug dissolution. Int. J. Pharm. 453, 12-24. [4] Nernst, W., 1904. Theorie der Reaktionsgeschwindigkeit in heterogenen Systemen. Z. Phys. Chem. 47, 52-55. [5] Brunner, E., 1904. Reaktionsgeschwindigkeit in heterogenen Systemen. Z. Phys. Chem. 47, 56-102. Page 3 3 Journal of Drug Delivery Science and Technology xxx (2015) 1e10 Contents lists available at ScienceDirect Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst Strategies for formulating and delivering poorly water-soluble drugs Marta Rodriguez-Aller, Davy Guillarme, Jean-Luc Veuthey, Robert Gurny* School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30, Quai Ernest Ansermet, 1211 Geneva 4, Switzerland a r t i c l e i n f o a b s t r a c t Article history: Received 19 March 2015 Received in revised form 12 May 2015 Accepted 12 May 2015 Available online xxx Water solubility is a key parameter in drug formulation since it highly influences drug pharmacokinetics and pharmacodynamics. In the past decades, the challenge with poorly water soluble drugs has been growing continuously. As a matter of fact, poorly soluble compounds represent 40% of the top 200 oral drugs marketed in the US, 33% of drugs listed in the US Pharmacopeia, 75% of compounds under development and 90% of new chemical entities. The present article presents and discusses the pharmaceutical strategies available to overcome poor water solubility in light of final drug product examples. First, chemical modifications based on the adjustment of the pH and the design of prodrugs are presented and discussed. Physical modifications based on modified solid states of the drug, small drug particles, cosolvents, surfactants, lipids and cyclodextrins are discussed in a second part. Finally, the option of modifying the route of administration is briefly presented. © 2015 Elsevier B.V. All rights reserved. Keywords: Insoluble drugs Formulation Delivery pH Pharmaceutical strategies Alternative administration route 1. Introduction The water solubility of drugs strongly influences their pharmacokinetics and pharmacodynamics and is a key parameter for formulators. Drug solubilization is based on the breaking of some drugedrug and waterewater interactions for the creation of new drugewater interactions. The strength of such interactions determines the solubility of a drug in water. Water solubility is one of the main parameters of the biopharmaceutical classification system (BCS) of drugs, as illustrated in Fig. 1A [1]. Moreover, “Lipinski's rule of 5” considers the solubility of drug candidates in view of the rejection of inappropriate candidates at early stages of the drug discovery process [2]. In the past decades, the challenges linked to poor water solubility have been continuously growing. The surge of combinatorial chemistry and high throughput miniaturized screening methods for drug discovery have resulted in an increase in molecular weight and lipophilicity of drug candidates [3e5]. In addition, the push towards increasing the potency of drugs often resulted in an increase in their lipophilicity (leading to stronger interactions with their receptors). Currently, poorly soluble compounds represent approximately 40% of the top 200 oral drugs marketed in the US and Europe, as shown in Fig. 1B [6]. In addition, they represent 90% * Corresponding author. E-mail address: [email protected] (R. Gurny). of new chemical entities, 75% of compounds under development and 33% of drugs listed in the US Pharmacopeia [2,3,6e11]. Interestingly, a variety of pharmaceutical strategies have been designed to address the formulation and delivery challenges presented by poorly soluble drugs, these are reviewed and discussed in the present article. 2. Strategies for formulating and delivering poorly watersoluble drugs The pharmaceutical strategies to address the poor water solubility of a drug can be organized into three categories according to the nature of the modification involved: the chemical, physical and administration strategies, as illustrated in Fig. 2. These approaches can of course be used separately or combined. Over the past decades, many efforts have been made to improve the formulation and delivery of poorly water-soluble immunosuppressants, prostaglandins and antineoplastic agents, which will often be used as examples in the following sections. It is worth mentioning that colloidal systems represent a more recent option for the formulation of poorly water soluble drugs that can involve chemical or physical modifications [12]. Thus, colloidal systems can be found in the sections describing prodrug design, small drug particles and surfactant-lipid-based formulations. The prodrug design can include drug-polymer nanoparticles and drug covalent link to inorganic nanoparticles. The use of small drug particles can involve nanocrystals and the use of nanoparticles for http://dx.doi.org/10.1016/j.jddst.2015.05.009 1773-2247/© 2015 Elsevier B.V. All rights reserved. Please cite this article in press as: M. Rodriguez-Aller, et al., Strategies for formulating and delivering poorly water-soluble drugs, Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.05.009 4 2 M. Rodriguez-Aller et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e10 Fig. 1. A) The biopharmaceutical classification system (BCS) and B) the solubility of the top 200 marketed oral drugs in the US and Europe (adapted from [6]). drug loading or adsorption. Finally, the surfactant and lipid formulations could include nanoemulsions, micelles, liposomes or solid lipid nanoparticles. 2.1. Chemical modifications 2.1.1. pH adjustment The pH can influence the solubility of a drug by affecting its degree of ionization as a function of its pKa. In its ionized form, a drug has a higher solubility than at its neutral form. However, drugs are generally neutral at physiological pH. Thus, the pH of the formulation can be adjusted with buffering excipients to ensure the presence of the most soluble form of the poorly water-soluble drug. For solid dosage forms, the buffering excipients control the pH of the microenvironment surrounding drug particles during in vivo dissolution [13]. Kranz and coworkers achieved a constant pHindependent release of the immunosuppressant, ZK811752, by adding organic acids to the final composition of the tablets [14]. The pH adjustment is a simple approach and represents a firstline strategy for the formulation of insoluble drugs. It is frequently Fig. 2. A schematic representation of the different strategies for formulating and delivering poorly water-soluble drugs. combined with other solubilizing approaches such as surfactants, cyclodextrins or cosolvents. The pH of the final formulation is selected not only according to drug solubility, but also considering its tolerance, bioavailability, efficacy and stability, which can strongly depend on the pH. In addition, the potential risk of drug precipitation after administration needs to be considered. 2.1.2. Prodrug design A prodrug can be defined as an inactive, chemically modified version of a parent drug displaying improved physico-chemical properties and being able to generate the active parent drug through a rapid biotransformation. Two main prodrug design categories can be identified: i) carrier-linked prodrugs where the parent drug backbone is covalently linked to a prodrug moiety and ii) bioprecursor prodrugs which are modified parent drugs with functional groups requiring hydration or redox reactions, as illustrated in Fig. 3A. In addition, pre-prodrugs or double prodrugs combine two prodrug design approaches in their design (carrierlinked and/or bioprecursor), one example being illustrated in Fig. 3B. The prodrug strategy has been gaining interest in the past years and today its usefulness in drug formulation is unquestionable. Prodrugs represent 10% of worldwide marketed drugs and were 33% of the small active molecules approved in 2008 [15,16]. Prodrug design represents a versatile and powerful approach that can solve a large variety of issues related to drug solubility, absorption, distribution, metabolism, toxicity or stability, among others [17,18]. The prodrug bioconversion is of major importance and needs to be carefully evaluated and optimized. Ideally, the prodrug should have an in vitro half-life one million times higher than its in vivo half-life. Such a difference is only possible with enzyme-based biotransformations [19]. For Anderson and Conradi, the prodrug of a poorly water soluble drug should not be limited to the covalent link of a promoiety to the parent drug, but should represent a new and optimized drug delivery system of its own [19]. In this sense, the use of prodrugs to address the challenges with poor water-solubility will be discussed through a number of examples, covering both the carrier and bioprecursor approaches. For carrier-linked prodrugs, the first type of prodrug design, four main carrier moieties can be used: i) hydrophilic groups, ii) hydrophobic groups, iii) amino acids and iv) macromolecules, as illustrated in Fig. 3A. Carrier-linked prodrugs are frequently used to simultaneously address the question of poor water-solubility of a drug and achieve its targeted delivery. The covalent linking of hydrophilic structures often confers a higher solubility to the parent drug. Phosphate ester prodrugs are Please cite this article in press as: M. Rodriguez-Aller, et al., Strategies for formulating and delivering poorly water-soluble drugs, Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.05.009 5 M. Rodriguez-Aller et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e10 3 Fig. 3. A) Prodrug design categories based on carrier-linked prodrugs and bioprecursors. B) Illustration of a pre-prodrug example combining the carrier-linked and bioprecursor designs. one of the most common examples for this group. Telzir® and Lexiva® (GlaxoSmithKline, Brentford, UK) contain fosamprenavir, the phosphate ester prodrug of the HIV protease inhibitor amprenavir. Fosamprenavir displays a water solubility 10 times higher than amprenavir as well as an increased bioavailability, allowing a simplification of the dosage regimen. With this prodrug formulation, the treatment went from 8 capsules twice a day to 4 tablets once a day, which has a direct impact on patient quality of life and compliance. Hydrophobic structures can also be used to improve the aqueous solubility of drugs. Their action is based on the disruption of some drugedrug interactions (e.g. H-bounds) resulting in a higher dissolution rate. The levodopa ethyl ester prodrug displayed a higher solubility and absorption than the parent levodopa, allowing its administration to Parkinson's disease patients as an oral solution instead of the conventional tablets with known absorption issues [20]. Interestingly, this levodopa ethyl ester is a double prodrug, levodopa being itself a prodrug of dopamine that targets the central nervous system. The development of novel formulations for the anticancer drug 5-fluorouracil (5-FU) based on the prodrug approach has been intensively investigated to increase its solubility, plasma half-life and selectivity. Xeloda® (HoffmannLaRoche, Basel, Switzerland) contains capecitabine, which is a double prodrug of 5-FU displaying an oral bioavailability close to 100% thanks to its high solubility, high absorption and low affinity for the intestine thymidine phosphatases [21]. Interestingly, for capecitabine design, hydrophobic hydrocarbon chains and amides were covalently linked to the doxifluridine backbone, which is already a pre-prodrug of 5-FU. After its oral absorption, capecitabine is biotransformed by carboxylesterases, deaminases and tumor-specific thymidine phosphorylases, releasing the cytotoxic 5-FU specifically in the tumor. Capecitabine combines the advantages of an enhanced oral availability with a tumor-specific activity [22e25]. The modifications with amino acids can simultaneously achieve two goals: increased water solubility and transporter-mediated absorption (using amino acid transporters). The diversity in physical properties of amino acids confers a high versatility to the approach. An interesting example is valacyclovir, the L-valyl ester prodrug of acyclovir marketed as Valtex® (GlaxoSmithKline, Brentford, UK). The bioavailability of valacyclovir is two times higher than acyclovir due to its higher solubility and active transport via amino acid receptors [26]. After intracellular absorption, valacyclovir is hydrolyzed, generating acyclovir, which requires activation by viral thymidine kinase and cellular kinases to finally inhibit herpes virus DNA polymerase. Valacyclovir can therefore also be considered a pre-prodrug. Finally, insoluble drugs can be combined with macromolecules. Drug-macromolecule conjugates can: i) assist drug solubilization, ii) decrease drug toxicity, iii) prevent drug degradation and iv) achieve drug targeting [27,28]. Hyaluronic acid, polyethylene glycol (PEG), hydroxypropylmetacrylamide (HPMA) and polyamidoamines or nitrodiol dendrimers are used in macromolecule prodrug designs. HPMA is a versatile tool for the formulation of poorly water-soluble drugs, such as anticancer agents (e.g. daunorubicine or wortmannin), and is especially well suited for being combined with drug targeting strategies [29e32]. Various HPMA conjugates (with doxorubicin, paclitaxel, camptothecin or palatinate) have gone through clinical trials [33]. In contrast to linear polymers, dendrimers have branched structures that allow the linkage of various drug molecules. They represent an interesting approach for the formulation and delivery of insoluble drugs. De Groot and coworkers presented nitrodiol-based dendrimers for the targeted release of the poorly water-soluble drug paclitaxel based on a single tumor specific activation that triggers a cascade of reactions towards paclitaxel release [34]. The triggering reaction can be designed to occur exclusively in the target tissue, allowing sitespecific drug release. Regarding bioprecursor prodrugs, the second type of prodrug design, an example is the Clinoril® (Merck, New Jersey, US) oral tablets, which contain sulindac, a non-steroidal anti-inflammatory drug [35,36]. Sulindac displays a 100-fold increased solubility and improved oral absorption compared to its parent drug [37,38]. The reduction of its sulphoxide group is necessary to generate the active sulphide form. The prodrug approach is therefore a powerful and versatile strategy to not only address issues with poor water solubility, but also to develop a myriad of strategies for efficient site-specific drug delivery. Nevertheless, the stability of prodrug formulations can be a hurdle since prodrugs require a high reactivity for a quick biotransformation, but also need an excellent stability for a long product shelf-life. 2.2. Physical modifications 2.2.1. Modified solid state Modifying the solid state of a drug influences the strength of drugedrug interactions, determining its solubility and dissolution Please cite this article in press as: M. Rodriguez-Aller, et al., Strategies for formulating and delivering poorly water-soluble drugs, Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.05.009 6 4 M. Rodriguez-Aller et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e10 rate. In general, a higher structural disorder in the solid leads to lower drugedrug interactions and a higher solubility. When using modified solid states of a drug, formulators need to find a tradeoff between: i) the potential increase in drug solubility, dissolution and/or availability and ii) potential stability issues. It is very important to ensure that the selected drug state is not altered during development, manufacturing and storage to guarantee that patients receive the appropriate form of the drug. Special attention needs to be paid to metastable polymorphs, salts, cocrystals and amorphous forms that tend to recrystallize into their most stable form. In addition, since the hydrated forms of a drug present a lower stability and solubility than its pure crystal, the risk of the drug form to be transformed into a hydrate needs to be evaluated. The different modified solid forms illustrated in Fig. 4 can be characterized as amorphous or crystalline (pure drug crystals, polymorphs, hydrates, salts and cocrystals) according to their disordered or ordered structures, respectively. Crystalline modified drug forms include polymorphs, hydrates, salts and cocrystals. Drug crystal polymorphs are composed solely of the pure drug, but different structures can lead to different drugedrug interactions and physico-chemical properties (e.g. density, melting point, conductivity, solubility or stability). Metastable polymorphs displayed a higher dissolution rate and solubility than the most stable polymorphs having a positive impact on therapeutic efficacy, as reported for cimetidine [39,40]. Besides, different polymorphs were demonstrated to have different degradation and thermodynamic profiles, some of which resulting in drug instability [41,42]. A comprehensive screening and monitoring of the potential polymorphic transformations during the drug life-cycle is mandatory to ensure drug quality, safety and efficacy [43]. Hydrates are crystalline drug structures that entrap water molecules, facilitating close packaging and drugedrug interactions. Generally, hydrates present limited pharmaceutical interest due to their lower solubility, bioavailability and stability compared to the anhydrous forms [41,44e47]. However, if a drug presents a high tendency to form hydrates, the study and characterization of the hydrated forms of the drug help to determine appropriate manufacturing, packaging and storage conditions (particularly important for hygroscopic drugs). Crystalline salts are based on a proton transfer between an ionizable group of the drug and a counter ion species. The resulting salt modifies the pH in the thin diffusion layer surrounding the drug, resulting in an increased solubility compared to the corresponding free form [48e50]. The counter ion species highly influences the solubility and must be carefully chosen [51]. Guzman and coworkers demonstrated the higher solubility and availability of the celecoxib sodium salt compared to its free form [52]. Cocrystals contain a drug and a cocrystal former, linked by Hbonds (different from salts in which a proton transfer takes place). These H-bonds decrease the strength of the drugedrug interactions compared to the pure drug crystal structure. The cocrystal approach has been successfully used for drug dissolution and bioavailability enhancement of poorly water soluble drugs [53e55]. This approach does not require the presence of ionisable groups on the drug. Amorphous solids are partially disordered solids where the drugedrug interactions are weaker than in the crystals. They are obtained either by preventing the formation of a crystalline structure or by disrupting an already existing crystal. Interestingly, amorphous forms can theoretically present solubilities up to 1600 times higher than crystalline forms [56]. The amorphization is usually combined with other small drug particle strategies such as solid dispersions (explained in the following “small drug particle” section). The solid dispersion of amorphous CsA particles in polymeric matrices was demonstrated to lead to a higher dissolution rate and an improved bioavailability compared to crystalline CsA [57,58]. In general, the solubility enhancement obtained with Fig. 4. Different drug solid forms that can be used in formulations, including the most stable pure drug crystal, as well as polymorphs, hydrates, salts, cocrystals and amorphous forms. Please cite this article in press as: M. Rodriguez-Aller, et al., Strategies for formulating and delivering poorly water-soluble drugs, Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.05.009 7 M. Rodriguez-Aller et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e10 amorphization is higher than that achieved with metastable polymorphs [11]. However, amorphous forms can have a higher hygroscopicity, reactivity and instability. 2.2.2. Small drug particles A decrease in particle size of poorly water-soluble drugs allows i) the increase of the drug surface area and its dissolution rate, ii) an improved bioavailability and iii) a reduced toxicity [58e69]. This approach can be combined with any of the modified solid states detailed above, cumulating the advantages of both strategies. Fine and ultrafine drug particles can be obtained using two types of strategies: reducing the size of preexisting drug particles or inducing drug solidification into small particles. The methods to reduce the size of preexisting drug particles are based on cuts, compression, impact, or attrition, or both impact and attrition [70]. The methods to form small drug particles are: hot-melt extrusion, hot-melt encapsulation, spray drying and supercritical fluid methods. It is also worth mentioning another approach consisting of the loading or superficial adsorption of poorly water-soluble drugs into nanoparticles [12,71]. Small drug particles form a metastable system that needs to be further stabilized to avoid agglomeration and crystalline growth. A large number of excipients can be used as stabilizers acting by electrostatic repulsion or steric stabilization (e.g. surfactants or polymers) [11,72,73]. Different systems can be formed depending on the environment surrounding the small particles: suspensions and nanosuspensions are obtained when drug particles are in a liquid environment, while solid dispersions are formed when the drug particles are embedded in a solid matrix. The advantages of using small drug particles for the formulation of poorly water-soluble actives are illustrated by the number of marketed products that employ this strategy. Three marketed formulations of the immunosuppressants sirolimus and everolimus used for the prevention of organ graft rejection can be cited as examples. Rapamune® (Pfizer, New York, US) is an oral tablet containing sirolimus nanocrystals (NanoCrystal® technology, Elan Drug Technologies, Dublin, Ireland). Solid dispersion strategies have been successfully used in the development of the everolimus formulations Certican® and Zortress® (Novartis, Basel, Switzerland). 2.2.3. Cosolvents A cosolvent is a water-miscible organic solvent used to increase the solubility of a drug in water. This approach is based on the theory that the dissolution is enhanced when the solute and solvent have similar physicochemical characteristics. The most important factor to be considered is the polarity of the mixture (i.e. its dielectric constant). A large variety of cosolvents such as ethanol, polyethylene glycol (PEG), propylene glycol (PG), glycerin, dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), dimethylsolfoxide (DMSO), as well as a number of oils (e.g. peanut, corn, sesame, olive or peppermint) can be used [73,74]. The cosolvent approach has been used for VePesid® (Bristol-Myers Squibb, New York, US) where the solubilization of the anticancer agent etoposide was achieved with a mixture of PEG 400, citric acid, glycerin and water. This cosolvent strategy presents some limitations linked to i) cosolvent taste and stability, ii) adverse physiological effects, iii) potential modification of the pharmacokinetic profile of the drug and iv) potential drug precipitation after administration. This strategy remains a simple option frequently used in combination with other solubilizing strategies for the formulation of poorly water-soluble drugs. Nevertheless, the risks of drug instability, drug precipitation and modification of the pharmacokinetic profile need to be considered. 5 2.2.4. Surfactants and lipids A surfactant is a surface-active agent that can stabilize interfaces. A large variety of surfactants can be listed, from nonionics (e.g. polyoxyethylene sorbitan fatty acid esters) to amphoteric (e.g. lecithins) and anionics (e.g. soaps, phospholipids). Cationic surfactants (e.g. quaternary ammonium) are less common due to toxicity issues and incompatibilities. Generally, a surfactant presents a polar “head” and an apolar “tail”. As illustrated in Fig. 5, a fraction of the surfactant adsorbs to the interfaces present in the system (liquid-air or liquideliquid interfaces), decreasing the interfacial tension and stabilizing the system. This property is used for emulsion stabilization. When the surfactant is added at a concentration above its critical micellar concentration, surfactant molecules self-assembly into micelles, liposomes or other structures [75]. Micelles presenting a hydrophilic spherical shell composed of polar heads and a hydrophobic core of apolar tails create an appropriate environment to solubilize poorly water-soluble drugs, as illustrated in Fig. 5A. Not all micelles are well suited for poorly water-soluble drugs, as it is the case of inverse micelles. Polymeric micelles deserve a special mention since they display interesting properties such as: i) low critical micellar concentration, ii) slow dissociation, iii) longer drug retention or potential increase in drug half-life [76e82]. Liposomes are globular bilayer formations allowing the solubilization of poorly water-soluble drugs inside the bilayer, as illustrated in Fig. 5B. Emulsions are dispersions of two immiscible phases (typically an oily phase and an aqueous phase) stabilized by a surfactant, as illustrated in Fig. 5C, and are appropriate for the formulation of lipophilic drugs [70]. The drug is solubilized in the oily phase, which could be the dispersed or dispersant phase, leading to oil-inwater (o/w) or water-in-oil (w/o) emulsions. Two types of emulsions can be identified: i) conventional emulsions and ii) microemulsions. A conventional emulsion is thermodynamically unstable needing an input of energy for its formation. Emulsion droplets have a diameter higher than 100 mm, conferring a milky aspect to the preparation. On the contrary, a microemulsion is thermodynamically stable and the droplets have a diameter between 6 and 80 nm, which does not affect optical transparency. The excipients and percentage of each phase play major roles in emulsion formation and stabilization. Some commonly used oily phases include vegetable oils, glycerols, fatty acids and their derivatives or glycerides. Microemulsions requiring further stabilization include a cosurfactant or a mixture of hydrophilic and lipophilic surfactants in specific proportions. Solid-lipid nanoparticles are another type of formulation based on surfactants and lipids, which combine the advantages of liposomes, emulsions and nanoparticles [12,83]. Surfactant-related formulations can be divided into three groups: i) a first group with ready-to-use formulations, ii) a second group with formulations requiring dilution in an aqueous vehicle prior to administration and iii) a third group involving “pro-formulations” that readily form the final emulsion or micelle system in contact with the biological medium. The presented groups are further discussed in light of examples of marketed products. Ready-to-use formulations include emulsions or micelle formulations intended to be administered as such. This is the case for Restasis® (Allergan, California, US) a cyclosporine A o/w emulsion developed for the topical treatment of dry eye syndrome. The formulations requiring a dilution in water prior to administration lead to the formation of: i) micelles, as in the case of Sandimmune® (Novartis, Basel, Switzerland) for the iv administration of cyclosporine A micelles, ii) liposomes, as for Visudyne® (Novartis, Basel, Switzerland) leading to verteporfin liposomes or iii) emulsions, as for Rapamune® (Pfizer, New York, US) leading to a Please cite this article in press as: M. Rodriguez-Aller, et al., Strategies for formulating and delivering poorly water-soluble drugs, Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.05.009 8 6 M. Rodriguez-Aller et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e10 Fig. 5. Surfactant distribution and self-assembly structures in aqueous environment for the formation of A) a micelle and B) a liposome and C) an emulsion droplet. cyclosporine A o/w emulsion for oral administration. “Pro-formulations” form their final appropriate system inside the biological medium. Micelles loaded with cyclosporine A are formed inside the gastrointestinal tract after the administration of Gengraf® (Abbott, Chicago, US). Emulsions can be obtained with self-emulsifying drug delivery systems (SEDDS), such as Neoral® and Sandimmune® (Novartis, Basel, Switzerland) leading to different cyclosporine A emulsions inside the gastrointestinal tract. Interestingly, Neoral was demonstrated to lead to a microemulsion with smaller droplet size than Sandimmune after selfemulsification, causing an increase in bioavailability of 239% and an increased plasma peak concentration (Cmax) and area under the curve of the plasma concentrationetime curve (AUC) when compared to Sandimmune [84e87]. The mechanisms behind selfemulsification have been identified as follows: i) diffusion and stranding, ii) osmotic pressure and iii) changes in the characteristics of the surrounding medium (e.g. pH or ionic strength) [88e92]. These surfactant-related formulations require extensive characterizations (e.g. solubility of the drug, size of the micelle, liposome or droplet, viscosity, osmolarity or stability) and can be related to the following risks: i) toxicity of the excipients (which could represent a high percentage of the final composition), ii) drug precipitation and iii) modification of the pharmacokinetic profile and biodistribution. 2.2.5. Complexation with cyclodextrins Cyclodextrins (CDs) are a family of cyclic oligosaccharides presenting a hydrophilic surface and a hydrophobic cavity that can be classified into a, b or g CDs according to their number of saccharide monomers. In addition to the “native non-substituted CDs”, a large variety of CD derivatives are also currently available. Fig. 6A illustrates the structure and characteristics of the four CDs included in the US Pharmacopeia [93]. CDs are known to act as drug solubilizers by forming dynamic inclusion complexes with poorly watersoluble drugs [94e100]. Despite the fact that the drug-CD complexation involves non-covalent interactions, it modifies the physico-chemical properties of both the CD and the drug. Complex formation and dissociation involve different mechanisms. Complex formation is linked to i) hydrophobic interactions, ii) release of the high-energy water molecules inside the CD and iii) “dissolution” of the drug inside the CD cavity. While drug-CD complex dissociation is based on the continuous dilutions and displacements that take place in vivo, leading to an efficient release of the drug from its complex, as shown in former investigations [95]. Besides an enhanced solubility, drug-CD complexes can lead to an improved drug availability by acting as drug carriers, and to an improved drug stability by preventing drug degradation [95,99,101e105]. The carrier-like behavior of CDs is illustrated in Fig. 6B. In addition, CDs are considered as non-toxic [103,104,106]. CD-based formulations require a comprehensive characterization regarding complex formation, stability and structure (e.g. complex stoichiometry, stability constants). The complexation approach was explored for the formulation of cyclosporine A with aCD resulting in a higher penetration, higher therapeutic effect and lower toxicity than the equivalent oily formulation [107e109]. However, to date, there is no commercial formulation containing cyclosporine A and CDs. In contrast, the combination of CDs and prostaglandins (PGs) can be found in a number of marketed formulations. For example, Caverjet Dual® (Pfizer, New York, US), Prostavasin® and Prostandin 500® (Ono, Osaka, Japan) contain PGE1 and aCD for the systemic treatment of cardiovascular-related diseases or dysfunctions. Prostarmon E® (Ono, Osaka, Japan) contains PGE2 and bCD for the induction of uterine contractions after its oral administration. The complexation strategy can also be combined with other solubilizing strategies such as i) hydrophilic polymers, ii) drug salts, iii) solid dispersion strategies or iv) cosolvents [102,110e115]. One example is Sporanox® (Janssen, New Jersey, US) which contains HPbCD together with propylene glycol as cosolvent for the formulation of itraconazole for the systemic treatment of fungal infections. The complexation strategy has been gaining interest in the past years, in conjunction with the surge of a variety of substituted CDs. This attractive and sophisticated technology brings new opportunities for the development and patent-protection of innovative formulations. Nevertheless, its cost is still high due to added regulatory hurdles and the elevated cost of materials. Please cite this article in press as: M. Rodriguez-Aller, et al., Strategies for formulating and delivering poorly water-soluble drugs, Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.05.009 9 M. Rodriguez-Aller et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e10 7 Fig. 6. A) Structure and characteristics of cyclodextrins (CDs) included in the US Pharmacopeia such as native aCD, bCD and gCD as well as the substituted hydroxypropylbCD (HPbCD) [93]. B) Potential effect of the use of a CD for the formulation of a drug that has to reach a biological barrier protected by an aqueous layer (such as mucosal secretion or tear fluid). The CD interacts with the drug by: i) forming a complex, ii) caring the drug across the aqueous layer and iii) allowing drug release close to the biological barrier for its subsequent absorption after complex dissociation. When the drug is formulated alone (upper part of the scheme), the crossing of the aqueous layer is more difficult and potentially a lower amount of drug can reach the biological barrier for its absorption. 2.3. Administration modification Another option to circumvent possible limitations of poorly water-soluble drug formulations is to modify the administration strategy. Although the oral route seems to be the golden standard for drug administration, it presents limitations linked to drug availability and potential systemic side effects. The use of an alternative administration route can be particularly interesting considering that previously mentioned physical or chemical modifications do not always allow an appropriate drug delivery. On one hand, alternative administration routes can be used to achieve a systemic release of a drug. Testosterone is a steroid hormone practically insoluble in water that has a rapid hepatic clearance, therefore requiring prolonged drug delivery systems or frequent administration. Intramuscular depot formulations or subcutaneous implants require invasive techniques for their administration. The transdermal and buccal routes were explored as alternative noninvasive options for the delivery of this drug. The developed transdermal and buccal formulations were demonstrated to achieve similar circulating concentrations of Fig. 7. Pros and cons of the presented strategies for formulating and delivering poorly water-soluble drugs. Please cite this article in press as: M. Rodriguez-Aller, et al., Strategies for formulating and delivering poorly water-soluble drugs, Journal of Drug Delivery Science and Technology (2015), http://dx.doi.org/10.1016/j.jddst.2015.05.009 10 8 M. Rodriguez-Aller et al. / Journal of Drug Delivery Science and Technology xxx (2015) 1e10 testosterone as the systemic options, while improving patient comfort [116,117]. On the other hand, local administration routes can be used for the treatment of local diseases. Indeed, local routes are the best option for the treatment of local diseases, allowing a high ratio of local to systemic drug concentrations which is translated into the achievement of the pharmacological effect with minimal systemic secondary effects. In this context, implants represent an attractive option for the local prolonged release of drugs; their main application fields are ophthalmology, cancer therapy and birth control [118]. Retisert® (pSivida Corporation, Watertown, US) is an intravitreal insert delivering active concentrations of flucinolone for up to three years allowing the treatment of chronic posterior uveitis [119]. Gliadel® (Arbor Pharmaceuticals, Atlanta, US) is an intracerebral implant containing carmustine placed on the brain tissue after surgical resection of the tumor for the prevention of glioblastoma recurrence [120]. Finally, Nuvaring® (Merck, New Jersey, US) is an intravaginal implant containing estrogens for contraceptive purposes which was demonstrated to result in a lower estrogen exposure than with the use of other oral or transdermal contraceptive options [121]. [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 3. Conclusion During the last decades, a number of pharmaceutical strategies have been developed for the formulation and delivery of poorly water-soluble drugs. In this article, eight of these strategies have been presented and critically discussed in light of examples of marketed products. The presented approaches included i) chemical modifications such as the adjustment of the pH and the design of prodrugs, ii) physical modifications such as the use of modified solid states of the drug, small drug particles, cosolvents, surfactants, lipids and cyclodextrins and iii) modifications of the administration strategy such as the use of alternative local administration approaches. These strategies can be used alone or in combination and offer a panel of options for formulators to address the challenges related to poorly water-soluble drugs. Pros and cons of each strategy are presented in Fig. 7. Through this article it could be seen that the development of a generic approach to solve drug solubility issues is not possible for two main reasons. The first reason is because each drug presents a different set of specific challenges. 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DRUG DEL. SCI. TECH., 23 (4) 403-408 2013 Amorphous drugs and dosage forms H. Grohganz1, K. Löbmann1, 2, P. Priemel1, 2, K. Tarp Jensen1, K. Graeser3, C. Strachan2, 4, T. Rades1* Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark 2 School of Pharmacy, University of Otago, Dunedin, New Zealand 3 Pharma Research and Early Development, Pharmaceutical Preformulation, F. Hoffmann-La Roche Ltd., Basel, Switzerland 4 Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland *Correspondence: [email protected] 1 The transformation to an amorphous form is one of the most promising approaches to address the low solubility of drug compounds, the latter being an increasing challenge in the development of new drug candidates. However, amorphous forms are high energy solids and tend to recrystallize. New formulation principles are needed to ensure the stability of amorphous drug forms. The formation of solid dispersions is still the most investigated approach, but additional approaches are desirable to overcome the shortcomings of solid dispersions. Spatial separation by either coating or the use of micro-containers has shown potential to prevent or delay recrystallization. Another recent approach is the formation of co-amorphous mixtures between either two drugs or one drug and one low molecular weight excipient. Molecular interactions between the two molecules provide an energy barrier that has to be overcome before single molecules are available for the formation of crystal nuclei, thus stabilizing the amorphous form. Key words: Amorphous – Solid dispersion – Glass solution – Spatial separation – Co-amorphous. In the pharmaceutical industry, poor physicochemical properties of potential new drug candidates such as low solubility, pose a challenge when turning a promising molecule into a successful drug. Low or erratic bioavailability can have its cause in low solubility or slow dissolution and therefore great efforts are taken in order to improve the solubility and subsequently the bioavailability of promising molecules. One approach has been to convert crystalline drug material into amorphous form. There are various techniques available to produce amorphous form such as spray-drying or freeze-drying, grinding, melt-extrusion or melt-quenching, and co-precipitation. Especially in the industry, techniques such as spray-drying and melt extrusion are commonly used to prepare amorphous material. A molecule in an amorphous form is in a higher energy state compared to its crystalline counterpart. Due to the increased mobility within the system, amorphous compounds exhibit a higher solubility which in turn may lead to a higher saturation concentration, thereby increasing the overall dissolution rate [1]. However, despite these theoretical expectations, there are limitations when working with an amorphous form. In the following sections, the solubility advantage, an overview on prediction of physical stability and a discussion on the existence of ‘the’ amorphous state will be briefly highlighted, followed by recent trends to improve the quality of amorphous drug formulations. The advantages of an amorphous drug form are to reach a higher solubility, create a supersaturated solution and effectively reach a higher bioavailability compared to its crystalline counterpart, due to the absence of a crystal lattice. In reality however, the expected behaviour is often difficult to observe [2], due to difficulties in determining the solubility of amorphous materials under true equilibrium conditions. When introducing an amorphous compound into an aqueous environment, there is an increased probability of recrystallisation, either directly from the amorphous material or through crystalline precipitation from the supersaturated solution. In recent reviews, practical issues of determining the actual advantages of using the amorphous state are discussed [3, 4]. Moisture acts as a plasticiser for amorphous compounds, reducing its glass transition temperature (Tg) and therefore enhancing recrystallisation, not only in direct contact with water, but also during storage through water in the gas phase. The physical stability and especially, the prediction thereof, is to date still an area of increased research as currently no method exists, to predict a priori the stability of an amorphous compound. Amorphous material above the Tg tends to crystallize fast, as sufficient mobility exists within the system to allow for nucleation and crystallisation of the material. It has long been hypothesised that the amorphous state below Tg does not possess sufficient mobility to allow for recrystallization, and it was proposed to store amorphous materials 50 K below its Tg to ensure stability [5]. However, this “rule of thumb” has been shown not to hold true as crystallisation was observed at temperatures well below Tg-50K [6]. I. Prediction of amorphous stability Ways of stabilising the amorphous state against recrystallisation and the prediction thereof has been at the centre of amorphous research in the past decades. As the glassy state is a non-equilibrium state, simple extrapolation from the stability under accelerated conditions based on the Arrhenius relationship do not apply, and real time storage experiments are currently the only way to confidently predict the stability of an amorphous form. Over the years, researchers have focused on correlating the stability from easily accessible measurements, thereby concentrating on thermodynamic factors, such as enthalpy and entropy, and on kinetic factors, such as the molecular mobility. Molecular mobility can be expressed as its reciprocal, the relaxation time, τ. Techniques like dielectric spectroscopy and solid state NMR can measure the relaxation of a sample directly, however, these techniques are not commonly available in every laboratory setting and measurements can be time consuming. A theoretical approach uses molecular dynamics simulations [7]. The mobility can also be indirectly assessed by using differential scanning calorimetry data (DSC) [8] and empirical approaches such as the Kohlrausch-Williams-Watts (KWW), Adam-Gibbs (AG) and the Vogel-Tammann-Fulcher (VTF) equations [9]. The calculated mobility is then correlated to observed physical stability, however the actual information content is limited, as only 2-3 drugs were actually used in these case study approaches. An excellent review on this topic has been published by Bhugra et al. [10]. 403 14 J. DRUG DEL. SCI. TECH., 23 (4) 403-408 2013 Amorphous drugs and dosage forms H. Grohganz, K. Löbmann, P. Priemel, K. Tarp Jensen, K. Graeser, C. Strachan, T. Rades Results showed that no simple method exists to easily correlate physical stability below the Tg with either molecular mobility or thermodynamic parameters for a larger set of drugs. This approach was refined by Baird et al. who used a set of 51 molecules and classified them according to their recrystallization tendency into 3 classes. Analyses of properties of the individual classes seemed to provide promising results [12]. Despite the unsatisfying results in correlating the physical stability of a number of different drugs with molecular mobility, it was observed that the relaxation time calculated by the KWW and the AG equations, could be used to rank the stability of two differently prepared amorphous forms of simvastatin [13]. These findings were followed up and confirmed for three differently prepared amorphous forms of indomethacin [14]. It was proposed that this method could be suitable to select the preparation technique that would give the most stable amorphous form for a given drug. Despite past research, the amorphous state is still not completely understood, and in the past only few amorphous products have been marketed. In the pharmaceutical industry, an amorphous form is rarely used on its own but rather formulated as a solid dispersion to enhance the stability. A new method has been introduced to prepare the amorphous state: the microprecipitated bulk powder technology (MBP) where the amorphous state is co-precipitated with a polymeric carrier to form an amorphous solid dispersion [15]. Recently the FDA approved a new anti-cancer drug from Roche, formulated as a solid dispersion using the novel MBP technology (Zelboraf). Figure 1 - Percentage amorphous content and relaxation time after 30 days of storage at Tg - 20 °C. (red triangles) Relaxation time, (grey bars) amorphous content. Error bars for the relaxation time lie within the symbol. Figure reproduced from [11] with permission from Elsevier. Graeser et al. published for the first time an approach where thermodynamic parameters as well as the molecular mobility were assessed and compared to the observed physical stability of a test set of 12 drugs (Figure 1) [11]. A modified version of the AG equation was used to calculate τ from DSC data, and DSC data was also used to calculate the differences in thermodynamic properties between the crystalline and the amorphous state (configurational properties). These configurational properties are strictly speaking only valid above the Tg, i.e. in the equilibrium supercooled melt state and the configurational entropy showed some correlation with the stability above the Tg (Figure 2). II. The influence of process parameters As stated above, the amorphous state can be produced by different techniques and the most common technique in the previous studies has been via the melt-quenching route. However, molecules that are thermo labile are often spray-dried or milled. Hence, the question arises, whether the manufacturing technique has an influence on the properties and stability behaviour of the amorphous state [16-18]. The influence of the preparation technique and parameter settings on the resulting amorphous state starting from both α-form and γ-form of indomethacin was investigated [19]. Using XRPD and Raman spectroscopy in combination with multivariate analysis, differences between the differently prepared amorphous forms on a molecular level could be detected (Figure 3). These molecular differences were related in differences in the physical stability during storage in the rank order of stability cryo-milled (γ) = ball milled (γ) < ball milled (α) < spray-dried < cryo-milled (α) < quench cooled. This ranking did not linearly correlate with the differences in molecular structure, and the authors indicated that these structural variations may not directly affect physical stability. Figure 2 - (a) Configurational entropy and enthalpy vs. stability of the drugs above Tg. (b) Configurational Gibbs free energy vs. stability. Stability was assessed by relating the recrystallization temperature to the glass transition and melting temperature via the reduced recrystallization temperature [(Tc - Tg)/(Tm - Tg)]. Figure reproduced from [11] with permission from Elsevier. Figure 3 - X-ray diffractograms of freshly prepared amorphous forms of indomethacin prepared by different preparative techniques. Figure reproduced from [19] with permission from Elsevier. 404 15 Amorphous drugs and dosage forms H. Grohganz, K. Löbmann, P. Priemel, K. Tarp Jensen, K. Graeser, C. Strachan, T. Rades J. DRUG DEL. SCI. TECH., 23 (4) 403-408 2013 Taking the same preparation method but varying the parameters, such as decreasing the cooling rates for QC material and prolonging the milling time also led to X-ray amorphous material with different quality. It was concluded that also different parameter settings within the same technique will result in different amorphous states. The differences in cooling rate were detectable on a molecular level; however, the dissolution behaviour was not affected. For the milled material however, differences in the dissolution were observed for the amorphous material and an increase compared to the crystalline material was only observed after exceeding a critical minimum milling time [19, 20]. III. Amorphous drug formulations The stabilisation of amorphous drugs is usually achieved by addition of a second substance. The approaches can be grouped depending whether the other compound is or is not interacting on a molecular level with the drug, and whether the substance is a polymer or a small molecule. With larger polymers, usually glass solutions are formed, where the drug substance is dissolved molecularly in the polymer or vice versa. At the University of Copenhagen two newer approaches are currently in the focus of investigation. Spatial separation is an approach where the second substance is in contact with the drug on a particular level, i.e. surrounded by microcontainer walls or by coatings of particles. The other approach is the formation of co-amorphous mixtures where the drug is stabilised via molecular interaction with another small molecule. Figure 4 - Crystalline content of amorphous indomethacin stored in microcontainers (174 and 223 μm) and in bulk phase at 23 % RH. Figure reproduced from Nielsen et al., 2012 with permission from Elsevier. 2. Spatial separation Another recent approach is to stabilise amorphous materials via spatial separation [35]. Indomethacin (IMC) was transformed to the amorphous form by quench cooling it into micro-containers (73, 174 and 223 μm diameter). Those were stored at 23 % RH and the physical stability of amorphous IMC was monitored with Raman spectroscopy and compared to bulk amorphous IMC. While around 50 % of the bulk material crystallised to the γ form after 5 days, the 174 μm microcontainers showed around 15 % and the 223 μm microcontainers around 25 % of crystallinity (see Figure 4). However, the 73 μm containers recrystallised fast to the α form which would be expected for storage condition above 42 % RH [36]. This observation was explained with the container material possibly absorbing water vapour or capillary condensation, both explanations would lead to a higher humidity in the direct environment of the amorphous IMC, hence resulting in the α form. Applying a coat on the surface of amorphous particles is another way of spatial separation. Amorphous IMC particles were coated with gold using a sputter coater or with polyelectrolytes via electrostatic assembly [37]. The physical barrier around the particles improved their physical stability by reducing surface mobility, and crystallites already existing before the coating process did not grow upon further storage [37]. Similarly, griseofulvin [38] and nifedipine [39] which both undergo surface crystallisation, showed reduced recrystallisation after a gold film was applied. Spatial separation/reduction of molecular mobility on the surface requires no functional groups such as H-bond acceptors and/or donors for stabilisation of amorphous drugs and should therefore be generally applicable to a broader range of drugs. However, it was also shown that the substance applied as coat also influences the physical stability of amorphous celecoxib (inulin < PVA ~ PVAP) [40]. DSC studies provided evidence for drug polymer mixing at the interface, hence stabilisation might be further improved through drug-polymer interactions in this case. 1. Glass solutions and solid dispersions Glass solutions are the most established systems to improve solubility and stabilize amorphous drugs [21-23]. Usually they consist of a drug and a polymer, often polyvinylpyrrolidone (PVP), polyvinylalcohol, cellulose derivates or polyethyleneglycole (PEG) and they can be prepared through melt quenching, ball milling, spray drying or hot melt extrusion [24]. Analytically the existence of a single glass transition temperature as well as peak shifts in infrared and Raman spectroscopy due to interactions are taken as indicator for the successful formation of a glass solution [25, 26]. However, it was shown that amorphous felodipine PVP solid dispersions with different PVP contents consisted of nano-domains of drug and polymer rather than being a molecularly dispersed system [27]. In another study a solid dispersion of a drug and PVP/VA was prepared via hot melt extrusion with two different sets of processing parameters [28]. While the data of freshly prepared samples differed neither in DSC nor XRPD, one sample already started to recrystallize after two months while the other was still amorphous. Raman microscopy revealed differences in the drug distribution in the less stable sample being less homogeneous. But even in a formulation with drug molecularly dissolved into the polymer, phase separation may occur during storage, especially when stored at high temperature and/or humidity. The strength of drug polymer interaction, hygroscopicity and API hydrophobicity were found to influence phase separation [29]. To be able to predict if a glass solution of a certain drug polymer pair would be successful and if phase separation would be likely to occur, the solubility/miscibility of drug and polymer were considered. The solubility parameters [30] and the Flory Huggins interaction parameter [31] have been calculated and good correlation to experimental data was found. In another approach [32] pair distribution functions (PDF) of single components and the drug (or sugar) polymer mixtures were compared to each other and differences were attributed to a glass solution formation and confirmed with DSC data (single Tg or two Tgs). The improved physical stability has not yet been clearly linked to a stabilisation mechanism, but is probably a complex result of several factors. In literature the increase of the Tg, antiplastisation, decreased molecular mobility of the drug molecules by the polymer chains [33], intermolecular interactions [34], type and ratio of polymer have been discussed as the main stabilising factors. 3. Co-amorphous drug formulations In the past few years interest towards the use of amorphous multicomponent mixtures comprising only molecules with a low molecular weight, instead of the use of large polymers, has increased. The use of small molecules, such as urea, citric acid, sugars and nicotinamide, as excipients in binary amorphous blends has been reported [41-44]. A stabilization effect of anhydrous citric acid on the amorphous form of paracetamol in binary mixtures was observed. The reason for the increased stability could be later assigned to hydrogen bonding between paracetamol and citric acid [45]. To further explore this approach using low molecular weight components, Chieng et al. developed co-amorphous drug/drug mixtures. 405 16 J. DRUG DEL. SCI. TECH., 23 (4) 403-408 2013 Amorphous drugs and dosage forms H. Grohganz, K. Löbmann, P. Priemel, K. Tarp Jensen, K. Graeser, C. Strachan, T. Rades The idea behind these systems was first, to create stable amorphous systems without the use of polymers, and second, developing new formulations of two drug candidates that could be used in combination therapy [46], for example a pharmacological relevant pair of drugs. In addition, the term “co-amorphous” was introduced for the first time to differentiate amorphous blends comprising only low molecular weight components to amorphous drug-polymer mixtures, which are generally referred to as solid dispersions or glass solutions. In their study, co-amorphous mixtures of indomethacin (IND) and ranitidine hydrochloride (RAN) in weight ratios of 2:1, 1:1 and 1:2 were produced by vibrational ball milling. They could show that the pure drugs alone were poor glass formers, i.e. it was not possible to transform them into the amorphous state under similar milling conditions. This changed drastically when mixtures of two components were processed. Fully co-amorphous blends were obtained and showed high physical stability against crystallization. Interestingly, the order of physical stability did not follow the order of increasing Tg as initially expected, but could be addressed to molecular interactions between IND and RAN at molecular and bulk level. These interactions were most distinctive in the co-amorphous 1:1 mixture. Thus, the 1:1 blend was more stable than the co-amorphous mixtures at 2:1 and 1:2 even though it had an intermediate Tg to those of the 2:1 and 1:2 mixtures. However, this finding could not be related to specific interactions in a 1:1 molar ratio since weight ratios were used in the study. In order to further study stability benefits of the 1:1 mixtures and potential molecular interactions, Allesø et. al. investigated coamorphous mixtures of naproxen (NAP) and cimetidine (CIM) at molar ratios instead of weight ratios [47]. Indeed, the co-amorphous blend at the molar ratio 1:1 again showed the highest physical stability in spite of its Tg being in between those of the co-amorphous mixtures at 2:1 and 1:2. The 1:1 blend remained amorphous up to approx. six month whereas the other mixtures showed recrystallization of the excess component (Figure 5). In addition, the 1:1 co-amorphous mixture showed a four-fold and two-fold increase in the intrinsic dissolution rate of naproxen and cimetidine, respectively, compared to the pure crystalline compounds. Furthermore, the drugs were released in a synchronized 1:1 molar fashion which means that with every NAP molecule one CIM molecule is released into the dissolution medium. These findings were explained by specific interactions between NAP and CIM in a pair-wise 1:1 molar fashion. The concept of co-amorphous drug/drug formulations was further investigated with respect to the factors influencing dissolution and stability in studies on co-amorphous IND/NAP [48] and glipizide (GPZ)/simvastatin (SVS) [49]. In the study on IND and NAP, we were able to confirm the result from the previous studies with respect to a synchronized release of the 1:1 molar blend and recrystallization of the excess component in the 1:2 and 2:1 molar ratios whereas the 1:1 ratio samples remained amorphous, even though the 1:1 molar ratio had an intermediate Tg to the 2:1 and 1:2 ratio mixtures. It was possible to show specific molecular interactions between the two drugs IND and NAP at the 1:1 molar ratio. In particular, the creation of a heterodimer between both drugs could be confirmed using FTIR spectroscopy and density functional theory (DFT) calculations [50]. The specific hydrogen bonds between IND and NAP stabilized the co-amorphous 1:1 blend (heterodimers). In order to convert back to the respective individual crystalline drugs, it would be necessary to break the hydrogen bond in the heterodimer, followed by reorientation of two like-molecules to form a homodimer. These homodimers would then be able to create crystal nuclei, which are the starting points for crystal growth. In this respect, the 2:1 and 1:2 mixtures contain two species of dimers, heterodimers between IND and NAP and homodimers between the molecules of the excess component. These homodimers are readily able to recrystallize. Therefore, the 1:1 molar ratio showed the highest physical stability among the mixtures investigated. It was concluded that specific molecular interactions between components in a co-amorphous formulation can offer the basis for an intrinsic resistance towards recrystallization. Overall, it was concluded that intermolecular interactions play a crucial role in co-amorphous formulations and have a high impact on the physical stability regardless the Tg. The study on co-amorphous mixtures between SVS and GPZ was special, in the sense that the blends were homogeneous one phase systems but did not show any signs of intermolecular interactions [49]. The dissolution of these mixtures revealed no advantage over that of the pure amorphous drugs. However, the storage stability of the co-amorphous mixtures was increased compared to the individual amorphous drugs and amorphous physical mixtures. It could be shown that molecular level mixing present in co-amorphous systems positively affected physical stability. Since not every drug has a suitable partner drug for combination therapy, there was a strong need to extend this approach to coamorphous mixtures comprising a drug and low molecular weight excipients. Amino acids exist in the biological receptor of drugs and those amino acids that are binding to the drug at the active site consequently interact strongly with drugs in vivo. Löbmann et al. [51, 52] introduced a strategy in which receptor amino acids relevant for a drug were chosen in order to create a physically stable co-amorphous systems (Figure 6). Co-amorphous blends were prepared by ball Figure 5 - XRPD patterns of physical stability study at various storage temperatures of co-amorphous NAP-CIM at the molar ratios 2:1, 1:1 and 1:2 showing the recrystallization of the excess component after 33 days. Figure reproduced from Allesø et al., 2009, with permission from Elsevier. Figure 6 - Schematic depiction of the research strategy using amino acids as co-amorphous excipients: a. find the active receptor site of a given drug (adapted from [53]), b. choose the drug binding amino acids, c. prepare a co-amorphous formulation, d. intrinsic dissolution testing. 406 17 Amorphous drugs and dosage forms H. Grohganz, K. Löbmann, P. Priemel, K. Tarp Jensen, K. Graeser, C. Strachan, T. Rades milling and contained either of the two drugs carbamazepine (CBZ) and IND in a combination with a corresponding receptor amino acid, i.e. phenylalanine (PHE)/ tryptophan (TRP)[53] and arginine (ARG)/ tyrosine (TYR)[54], respectively. It was possible to obtain homogeneous co-amorphous blends at a 1:1 molar ratio containing either one of the two drugs and the amino acids ARG, PHE and TRP. The dissolution rate of these mixtures was increased over the respective crystalline and amorphous pure drugs and the co-amorphous formulations showed excellent physically stability. The improved physical stability could be explained by markedly higher Tgs compared to the individual drugs and molecular level mixing with the amino acids. Studying interactions revealed that in all of the co-amorphous mixtures with CBZ, both H-bonding and π-π interactions could be identified. It could be shown that interactions play a crucial role in the physical stability of co-amorphous mixtures as the co-amorphous mixtures with molecular interactions in this study also show the best physical stability kinetics. Overall molecular interactions between drug and amino acids were only identified in those co-amorphous mixtures that included at least one amino acid from the biological target site as initially anticipated. However, the interactions included the side chains, the carboxylic acid and amino functional groups of the amino acids which are involved in the peptide bond in the backbone of the protein and thus not available for specific interactions with the drug target at the biological active site. Therefore, a direct connection between the interactions occurring between the drugs and the amino acids in the co-amorphous mixtures and the biological targets could not be made. Formulating co-amorphous blends with a drug and amino acids from the natural binding site, however, can give a good starting point in choosing potential amino acids for a co-amorphous formulation. In order to identify the most advantageous amorphous drug formulations the choice of amino acids as excipients should be based on an assessment of Tg, molecular interactions as well as molecular level mixing. Amino acids were shown to be a promising alternative to solid dispersions of drug in polymers. The use of small molecular amino acids has a potential in reducing the bulk volume compared to formulations based on solid dispersions, as large quantities of polymer are required to create a solid solution. Furthermore, limited solubility of some drugs in the polymers as well as high risk of recrystallization due to hygroscopicity of most polymers are challenges, which co-amorphous formulations including amino acids could overcome. In addition, amino acids are non-toxic and generally regarded as safe. All together amino acids have a potential to form the basis for a new platform technique to overcome challenges associated with the amorphous state of poorly soluble drugs. J. DRUG DEL. SCI. TECH., 23 (4) 403-408 2013 gaining attention. Here suitable processes that enable the formation of a coat without triggering recrystallisation of the drug will need to be investigated in future. Last but not least, the concept of coamorphous mixtures is a new rationale for the stabilisation of amorphous compounds, mostly based on molecular interactions with other small molecular compounds. New methods to produce co-amorphous mixtures in industrial scale and the way to find or design the optimal excipient for each specific drug need to be investigated to reach the full potential of this approach. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. * 13. Amorphous systems will gain in importance for the pharmaceutical industry in future, due to an increasing number of poorly water soluble drug candidates. A transfer of the crystalline drug to its amorphous counterpart is often a way to increase the solubility. However, it has been shown that a defined single amorphous state does not exist, and that both the type of production process as well as its process parameters influence the molecular order of the amorphous compound. In order to stabilise the obtained amorphous drug, different formulation strategies can be followed. While the application of solid solutions is the most established approach, newer approaches are developed to handle its shortcomings, such as phase separation upon storage or the use of large amounts of excipients. Spatial separation by microcontainers can avoid the phase separation phenomena, however the prevention of water adsorption due to the large surface of the containers as well as the filling procedure of the containers need to be optimised. 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Vasconcelos T., Sarmento B., Costa P. - Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. - Drug Discov. Today, 12 (23-24), 1068-1075, 2007. Leuner C., Dressman J. - Improving drug solubility for oral delivery using solid dispersions. - Eur. J. Pharm. Biopharm., 50 (1), 47-60, 2000. Patterson J.E., James M.B., Forster A.H., Lancaster R.W., Butler J.M., Rades T. - Preparation of glass solutions of three poorly water soluble drugs by spray drying, melt extrusion and ball milling. - Int. J. Pharm., 336 (1), 22-34, 2007. Tobyn M., Brown J., Dennis A.B., Fakes M., Gao Q., Gamble J., Khimyak Y.Z., McGeorge G., Patel C., Sinclair W., Timmins P., Yin S. - Amorphous drug-PVP dispersions: Application of theoretical, thermal and spectroscopic analytical techniques to the study of a molecule with intermolecular bonds in both the crystalline and pure amorphous state. - J. Pharm. Sci., 98 (9), 3456-3468, 2009. Taylor L.S., Zografi G. - Spectroscopic characterization of interactions between PVP and indomethacin in amorphous molecular dispersions. - Pharm. Res., 14 (12), 1691-1698, 1997. Karavas E., Georgarakis M., Docoslis A., Bikiaris D. - Combining SEM, TEM, and micro-Raman techniques to differentiate between the amorphous molecular level dispersions and nanodispersions of a poorly water-soluble drug within a polymer matrix. - Int. J. Pharm., 340 (1-2), 76-83, 2007. Qian F., Huang J., Zhu Q., Haddadin R., Gawel J., Garmise R., Hussain M. - Is a distinctive single Tg a reliable indicator for the homogeneity of amorphous solid dispersion? - Int. J. Pharm., 395 (1-2), 232-235, 2010. Rumondor A.F., Wikström H., Van Eerdenbrugh B., Taylor L. - Understanding the tendency of amorphous solid dispersions to undergo amorphous-amorphous phase separation in the presence of absorbed moisture. - AAPS PharmSciTech, 12 (4), 1209-1219, 2011. Greenhalgh D.J., Williams A.C., Timmins P., York P. - Solubility parameters as predictors of miscibility in solid dispersions. - J. Pharm. Sci., 88 (11), 1182-1190, 1999. Marsac P., Shamblin S., Taylor L. - Theoretical and practical approaches for prediction of drug-polymer miscibility and solubility. - Pharm. Res., 23 (10), 2417-2426, 2006. Newman A., Engers D., Bates S., Ivanisevic I., Kelly R.C., Zografi G. - Characterization of amorphous API:Polymer mixtures using X-ray powder diffraction. - J. Pharm. Sci., 97 (11), 4840-4856, 2008. Van den Mooter G., Wuyts M., Blaton N., Busson R., Grobet P., Augustijns P., Kinget R. - Physical stabilisation of amorphous ketoconazole in solid dispersions with polyvinylpyrrolidone K25. - Eur. J. Pharm. Sci., 12 (3), 261-269, 2001. Janssens S., Van den Mooter G. - Review: physical chemistry of solid dispersions. - J. Pharm. Pharmacol., 61 (12), 1571-1586, 2009. Nielsen L.H., Keller S.S., Gordon K.C., Boisen A., Rades T., Mullertz A. - Spatial confinement can lead to increased stability of amorphous indomethacin. - Eur. J. Pharm. Biopharm., 81 (2), 418-425, 2012. Andronis V., Yoshioka M., Zografi G. - Effects of sorbed water on the crystallization of indomethacin from the amorphous 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. state. - J. Pharm. Sci., 86 (3), 346-351, 1997. Wu T., Sun Y., Li N., de Villiers M.M., Yu L. - Inhibiting surface crystallization of amorphous indomethacin by nanocoating. Langmuir, 23 (9), 5148-5153, 2007. Zhu L., Jona J., Nagapudi K., Wu T. - Fast surface crystallization of amorphous griseofulvin below Tg. - Pharm. Res., 27 (8), 1558-1567, 2010. Zhu L., Wong L., Yu L. - Surface-enhanced crystallization of amorphous nifedipine. - Mol. Pharm., 5 (6), 921-926, 2008. Puri V., Dantuluri A.K., Bansal A.K. - Barrier coated drug layered particles for enhanced performance of amorphous solid dispersion dosage form. - J. Pharm. Sci., 101 (1), 342-353, 2012. Ahuja N., Katare O.P., Singh B. - Studies on dissolution enhancement and mathematical modeling of drug release of a poorly water-soluble drug using water-soluble carriers. - Eur. J. Pharm. Biopharm., 65 (1), 26-38, 2007. Descamps M., Willart J.F., Dudognon E., Caron V. - Transformation of pharmaceutical compounds upon milling and comilling: the role of Tg. - J. Pharm. Sci., 96 (5), 1398-1407, 2007. Masuda T., Yoshihashi Y., Yonemochi E., Fujii K., Uekusa H., Terada K. - Cocrystallization and amorphization induced by drug-excipient interaction improves the physical properties of acyclovir. - Int. J. Pharm., 422 (1-2), 160-169, 2012. Hoppu P., Jouppila K., Rantanen J., Schantz S., Juppo A.M. Characterisation of blends of paracetamol and citric acid. - J. Pharm. Pharmacol., 59 (3), 373-381, 2007. Schantz S., Hoppu P., Juppo A.M. - A solid-state NMR study of phase structure, molecular interactions, and mobility in blends of citric acid and paracetamol. - J. Pharm. Sci., 98 (5), 18621870, 2009. Chieng N., Aaltonen J., Saville D., Rades T. - Physical characterization and stability of amorphous indomethacin and ranitidine hydrochloride binary systems prepared by mechanical activation. - Eur. J. Pharm. Biopharm., 71 (1), 47-54, 2009. Allesø M., Chieng N., Rehder S., Rantanen J., Rades T., Aaltonen J. - Enhanced dissolution rate and synchronized release of drugs in binary systems through formulation: Amorphous naproxen-cimetidine mixtures prepared by mechanical activation. - J. Control. Release, 136 (1), 45-53, 2009. Löbmann K., Laitinen R., Grohganz H., Gordon K.C., Strachan C., Rades T. - Coamorphous drug systems: enhanced physical stability and dissolution rate of indomethacin and naproxen. Mol. Pharm., 8 (5), 1919-1928, 2011. Löbmann K., Strachan C., Grohganz H., Rades T., Korhonen O., Laitinen R. - Co-amorphous simvastatin and glipizide combinations show improved physical stability without evidence of intermolecular interactions. - Eur. J. Pharm. Biopharm., 81 (1), 159-169, 2012. Löbmann K., Laitinen R., Grohganz H., Strachan C., Rades T., Gordon K.C. - A theoretical and spectroscopic study of coamorphous naproxen and indomethacin. - Int. J. Pharm., doi: 10.1016/j.ijpharm.2012.05.016 (0), 2012. Löbmann K., Grohganz H., Laitinen R., Strachan C.J., Rades T. - Amino acids as co-amorphous stabilisers for poorly water soluble drugs - Part 1: Preparation, stability and dissolution enhancement. - Eur. J. Pharm. Biopharm., under revision 2013. Löbmann K., Laitinen R., Strachan C.J., Rades T., Grohganz H. - Amino acids as co-amorphous stabilisers for poorly water soluble drugs - Part 2: Molecular Interactions. - Eur. J. Pharm. Biopharm., under revision 2013. Rowlinson S.W., Kiefer J.R., Prusakiewicz J.J., Pawlitz J.L., Kozak K.R., Kalgutkar A.S., Stallings W.C., Kurumbail R.G., Marnett L.J. - A novel mechanism of cyclooxygenase-2 inhibition involving interactions with Ser-530 and Tyr-385. - J. Biol. Chem., 278 (46), 45763-45769, 2003. Yang Y.-C., Huang C.-S., Kuo C.-C. - Lidocaine, carbamazepine, and imipramine have partially overlapping binding sites and additive inhibitory effect on neuronal Na+ channels. - Anesthesiology, 113 (1), 160-174 2010. Manuscript Received 7 March 2013, accepted for publication 30 March 2013. 408 19 new optiform‰ solution suite enhanced bioavailability in 12 weeks! easier Integrated solution at one simple price with minimal API needed. simpler Optimal recommendations based on real data from a dedicated scientific advisor. faster Accelerated parallel process with 4 superior technologies allowing for optimized animal pK prototypes in 12 weeks! ˝ 2015 Catalent Pharma Solutions. All rights reserved. Rigorous science. Superior technologies. From molecule to dose form. Learn more at catalent.com/optiform us + 1 888 SOLUTION eu 00800 8855 6178 [email protected] 20 J. DRUG DEL. SCI. TECH., 23 (4) 375-382 2013 Recent developments in oral lipid-based drug delivery N. Thomas, T. Rades, A. Müllertz* Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark *Correspondence: [email protected] The increasing number of poorly water-soluble drugs in development in the pharmaceutical industry has sparked interest in novel drug delivery options such as lipid-based drug delivery systems (LbDDS). Several LbDDS have been marketed successfully and have shown superior and more reliable bioavailability compared to conventional formulations. However, some reluctance in the broader application of LbDDS still appears, despite the growing commercial interest in lipids as a drug delivery platform. This reluctance might at least in part be related to the complexity associated with the development and characterization of LbDDS. In particular, the lack of standardized test protocols can be identified as the major obstacles for the broader application of LbDDS. This review seeks to summarize recent approaches in the field of lipid-based drug delivery that try to elucidate some critical steps in their development and characterization. Key words: Lipids – (super-)SNEDDS – In vitro lipolysis – Absorption. The oral route is considered the preferred way of drug administration since it has proven to be convenient and acceptable for the majority of patients [1]. Moreover, oral dosage forms are relatively easy to manufacture at comparably low costs, making them an attractive delivery form for the pharmaceutical industry [2]. However, conventional oral delivery is currently facing challenges by an increasing number of poorly water-soluble drugs demonstrating poor and variable absorption. The unmet need for reliable and reproducible absorption of many poorly water-soluble compounds has stimulated formulation scientists to utilize enabling drug delivery systems that differ greatly from conventional delivery systems [3]. Amongst other approaches the utilization of lipidbased drug delivery systems (LbDDS) has attracted particular interest, not only in academia but also in pharmaceutical industry, as indicated by the number of LbDDS approved for the market (Table I). However, the number of commercially available LbDDS is still limited indicating that their full potential still remains to be explored. Most likely the reluctance of the pharmaceutical industry to develop LbDDS as a platform delivery system for (lipophilic) poorly water-soluble drugs is at least in part due to the complexity of LbDDS and the current lack of in vitro protocols predictive for the performance of LbDDS in vivo. On the other hand there are also economical considerations such as greater cost of goods for the development and formulation of LbDDS compared to conventional solid dosage forms. As the majority of LbDDS are liquids, their manufacturing requires expertise in hard-gelatine capsule filling or soft-gel encapsulation technology which might not be readily available in-house [3, 4]. Furthermore, compared to solid dosage forms, the existence of the drug dissolved in a LbDDS poses a potential disadvantage with regard to the chemical stability and shelf-life for the intended drug product [5, 6]. The current work reviews the possibilities and limitations of LbDDS to improve the bioavailability of lipophilic drugs and describes the challenges and caveats in the development and in vitro characterization of LbDDS, highlighting the importance to identify the solid state properties of drug precipitating during in vitro lipolysis. Together with the evaluation of different animal models used during recent in vivo studies this review aims at the better understanding of LbDDS for a broader application of these promising delivery systems. Table I - Examples of marketed LbDDS and their composition (adapted from reference [7]). Drug Trade name Lipid components Surfactants Hydrophilic cosolvents Alfacalcidol One-Alpha Sesame oil - - Amprenavir Agenerase - TPGS PEG 400, PG Ciprofloxacin Cipro Mediumchain TAG, lecithin Tween 20 - Clomethiazole edisilate Heminevrin Fractionated TAG of coconut oil - - Cyclosporin A Neoral MAG, DAG and TAG of corn oil Cremophor RH 40 Ethanol, glycerol, PG Cyclosporin A Sandimmune Corn oil Labrafil M2125CS Ethanol, glycerol Dronabinol Marinol Sesame oil - - Fenofibrate Fenogal - Gelucire 44/14 - Lopinavir/ ritonavir Kaletra - Cremophor RH 40 Ethanol, glycerin, PG Ritonavir Norvir Oleic acid Cremophor EL Ethanol Saquinavir Fortovase Mediumchain MAG and DAG - - Abbreviations: TPGS, tocopherol polyethylene glycosuccinate; PEG, polyethylene glycol; PG, propylene glycol; TAG, triacylglyceride; MAG, 2-monoacylglyceride; DAG, diacylglyceride. are associated with poor and variable absorption [8, 9]. Moreover, the absorption of poorly water-soluble drugs is often affected by the consumption of food [10]. In many cases absorption of poorly watersoluble drugs is facilitated by the presence of food. Lipids, in particular, have been recognized to play a profound role in the solubilization and absorption of lipophilic drugs. In an attempt to harness the beneficial I. The role of lipids in drug delivery Sufficient aqueous solubility along with good intestinal permeability is crucial for adequate drug absorption, ultimately leading to sufficient bioavailability [8]. Conversely, poorly water-soluble drugs 375 21 Recent developments in oral lipid-based drug delivery N. Thomas, T. Rades, A. Müllertz J. DRUG DEL. SCI. TECH., 23 (4) 375-382 2013 effects of lipids on drug absorption, research on LbDDS has become increasingly popular as a potential platform for the delivery of poorly water-soluble, lipophilic drugs [5, 7, 10, 11]. LbDDS include simple lipid solutions, liposomes, micellar solutions and more complex delivery systems such as self-emulsifying drug delivery systems (SEDDS) and self-nanoemulsifying drug delivery systems (SNEDDS). The common theme of these formulations is that a co-administered drug is presented in a dissolved state, thereby avoiding the critical dissolution step necessary for crystalline, poorly water-soluble drugs. While simple LbDDS may only comprise of a single lipid, SEDDS and SNEDDS typically contain four and more excipients (lipid, surfactant, co-surfactant, and cosolvent) forming anhydrous isotropic pre-concentrates. Following gentle agitation in aqueous medium the pre-concentrates generate kinetically stable emulsions (SEDDS, particle size 0.2-2 µm) or nano-emulsions (SNEDDS, particle size < 0.2 µm). In this context it should be mentioned that the term SNEDDS is preferred over SMEDDS (self-microemulsifying drug delivery systems) since the latter term should be reserved for thermodynamically stable systems [12, 13]. The rather loose use of the terminology might be attributed to the macroscopically similar emulsification process and the translucent to opaque appearance of the dispersions owing to their submicron particle sizes. In contrast to conventional excipients used for oral solid dosage forms, lipid excipients are susceptible to digestion in the gastrointestinal tract. In fact, digestion can be described as the first step after administration and dispersion of the LbDDS in a series of complex events often resulting in improved drug solubilization by colloidal structures such as vesicles and mixed micelles [14, 15]. Digestion of triacylglycerols (derived either from LbDDS or food) is initiated in the stomach by the hydrolytic activity of gastric lipase and contributes to approximately 10-20 % of the total lipolysis [16]. The surface active digestion products (diglycerides, partially ionized fatty acids) and the grinding forces of the stomach support the initial, crude emulsification of the chyme which subsequently empties into the duodenum. The following hydrolysis of the lipids by the concerted action of pancreatic lipase, co-lipase and bile salts finalizes the enzymatic breakdown of the lipids, generating two moles of fatty acids and one mole of 2-monoglycerides for each mole triacylglyceride (Figure 1). Many physiological and biochemical reactions of the digestion process have been described in great detail [18-21]. Rather simple oil emulsions were used in these pioneering studies compared to the complex LbDDS commonly employed for recent pharmaceutical applications. Nevertheless, the early investigations on lipid digestion provided important information such as that the digestion of fat emulsions depends on their initial physico-chemical properties (e.g. faster digestion of fine emulsions with smaller droplet size compared to coarse emulsions) providing the bases for later, pharmaceutical studies [22]. However, there is still a considerable lack in the understanding of how the digestion products interact with drugs leading to enhanced absorption. Many of the current approaches to understand drug absorption are concerned with the importance of drug solubilization in different biorelevant media using various levels of bile salt and phospholipids [23, 24]. While this approach appears feasible for the assessment of those drugs whose solubilities solely depend on the overall concentration of surfactant, the type of surfactant and the presence of digestion products can cause considerable deviation from the solubilities for other drugs [25-27]. As an example, danazol solubility in biorelevant media was not affected by the type of surfactant used, whereas fenofibrate and cinnarizine showed a substantial increase in their solubility in media supplemented with oleic acid/monoolein as model digestion products [27]. In addition to solubilization, several other effects of lipid excipients in LbDDS may contribute to enhanced drug absorption. For example highly lipophilic compounds will be directed towards the lymphatic route when administered with long-chain lipids [28, 29]. In particular, drugs that demonstrate pharmacological action in the lymphatic system might benefit from this route, as well as compounds susceptible to a high first pass metabolism [30, 31]. Moreover, the drug permeability can be elevated by LbDDS and their digestion products by the opening of tight junctions, inhibitory effects on efflux transporters and modulation of metabolic enzymes [30, 32, 33]. As an example, Risovic et al. found that glyceryl monooleate decreased the expression of P-gp protein and stimulated the intestinal lymphatic uptake of amphotericin B resulting in increased drug absorption [32]. The presence of lipids can also prolong the residence time of undissolved drug, for example from normal tablet formulations, in the gastrointestinal tract, which allows for a longer time for a drug to dissolve. It has been shown that relatively small amounts of lipids administered as LbDDS can induce effects comparable to those observed with dietary lipids [34]. Whilst complete dissolution is desirable for conventional formulations and compounds for which absorption is limited by dissolution, it is not relevant for LbDDS since the drug is usually already presented in a dissolved state. However, the importance of presenting the drug in a dissolved state requires further investigation in light of a recent study that found the same bioavailability of danazol administered either as a solution or as a suspension administered in the same lipid vehicle [35]. This data demonstrates that the absence of a dissolution step alone cannot account for the biopharmaceutical advantages of LbDDS, but that drug dissolution (in case of a suspension), solubilization and absorption from LbDDS are highly dynamic and complex. In fact, the underlying mechanisms determining the absorption of a co-administered drug from LbDDS are still not completely understood. However, several steps have been proposed to attain successful drug absorption. It is necessary that the drug is released from the formulation before it can be absorbed in the small intestine as neither micelles nor oil droplets can be absorbed intact through the intestinal epithelium [36]. According to this model, drug release from LbDDS can proceed in two ways: - the drug partitions directly from the formulation into the bulk (i.e. as free drug or solubilized in bile salt micelles) and subsequently into the enterocyte, or the drug is released from the formulation into the bulk upon the degradation of the formulation [11]. The first pathway has also been termed “interfacial partition” and can be considered as the only release mechanism for formulations devoid of digestible excipients [37, 38]; - the second pathway is restricted to formulations containing digestible excipients. These can be enzymatically degraded by pancreatic lipase and co-lipase, or other pancreatic esterases [7, 39]. The presence of exogenous lipids further stimulates the contraction of the gall bladder releasing bile salts and lecithin into the intestine. Together with Figure 1 - Illustration of pancreatic lipolysis at the lipid/water interface. The lipolytic activity of the pancreatic lipase alone (i) is largely decreased compared to the pancreatic lipase/co-lipase complex (ii). The structure of some surfactants and the accumulation of digestion products at the substrate surface inhibit the activity of the pancreatic lipase/co-lipase complex (iii). Orogenic displacement of these amphiphiles by bile salts re-establishes the activity of the pancreatic lipase/co-lipase complex (iv). Moreover, bile salts solubilize the digestion products by incorporation in mixed micelles and vesicles making them available for absorption through the intestinal wall (v). Reprinted from reference [17] with permission of Elsevier. 376 22 Recent developments in oral lipid-based drug delivery N. Thomas, T. Rades, A. Müllertz J. DRUG DEL. SCI. TECH., 23 (4) 375-382 2013 these endogenous solubilizing agents the degradation products of the formulation (fatty acids and 2-monoglycerides) produce a range of colloidal species [40-42] which may have a positive or negative influence on the solubilization capacity for the administered drug, depending on the nature of the drug, the LbDDS and the time course of digestion [35, 43-46]. Table II - Examples of frequently used excipients for the formulation of lipid-based drug delivery systems. Adapted from reference [47]. II. Development of lipid-based drug delivery systems The vast number of commercially available excipients (Table II) gives rise to a plethora of possible combinations for LbDDS [47]. It is perhaps due to this variety that the attempts for a classification system for LbDDS have not been able to fully accommodate their different physicochemical characteristics that could serve as a guide for formulation scientists [7, 48]. Only a few structured guidelines for the adequate selection of LbDDS for poorly water-soluble drugs have been proposed to date [4, 49]. However, the development of LbDDS is still largely a process driven by empirics rather than rational decision making. The proposed guidelines for the development of LbDDS start with the solubility assessment of a drug in a range of excipients and mixtures thereof. This is accompanied by the determination of pseudo-ternary phase diagrams to identify areas of mutual miscibility of selected excipients and areas of self-emulsification in case a self-emulsifying LbDDS is intended. Since the presence of drug can alter the emulsification process the drug should be already included in these initial studies [45]. The perspective for the successful formulation of a poorly watersoluble drug in a LbDDS is promising for drug candidates limited by solubility and dissolution rate rather than permeability, i.e. drugs belonging to Class 2 (poor solubility/good intestinal permeability) according to the Biopharmaceutics Classification System (BCS) [50]. However, it would be erroneous to expect all poorly water-soluble drugs respond equally well to the formulation as LbDDS. In fact, for drugs demonstrating a high degree of crystallinity, formulation as LbDDS is likely to fail if the drug is not lipophilic, as reflected by a low solubility in triglycerides and a low partition coefficient (logP < 2) [48]. Alternative formulation approaches for these hydrophobic drugs are reviewed elsewhere and include salt formation, particle size reduction, formulation as solid dispersions, and the use of cyclodextrins [8, 49, 51, 52]. In contrast, LbDDS are a likely formulation option for lipophilic compounds with logP > 4 and appropriate solubility of the drug in triglycerides [48]. If necessary, the drug’s solubility in LbDDS can be improved by the inclusion of surfactants, mixed glycerides, and cosolvents [7, 48]. It should be noted that the commonly used logP as an estimate for lipophilicity varies with pH for ionizable drugs. In order to account for this pH-dependency the distribution coefficient (logD) should be assessed for weak acids and bases at different pH values. As an example, the logD of the antimalarial drug halofantrine (a weak base) increased by three units when the pH was changed from pH 2 to 7 [53]. Importantly, the bioavailability of very lipophilic compounds (logP > 6) with a solubility in triglycerides greater than 50 mg/g can increase substantially by stimulation of the lymphatic absorption pathway, particularly when long-chain lipids are employed containing fatty acids with more than twelve carbons [53, 54]. The characterization techniques employed following the dispersion of the pre-concentrates in a dissolution paddle apparatus or simple magnetic stirrer include nephelometry [55], photon correlation spectroscopy [6, 38, 56], electron microscopy [40, 45, 57, 58], and recently, ultrasonic resonator technology [13, 59]. While the selection of SNEDDS is often based on a good dispersion performance (indicated by small polydispersity index and particle size) it is interesting to note that only few studies have found evidence that finely dispersing LbDDS are associated with improved performance in vivo [58, 60, 61]. The conventional screening of SNEDDS involves time consuming preparation and analyses of a considerable number of samples. Water-insoluble excipients Surfactants Cosolvents Beeswax Corn oil Glyceryl monooleate Ethanol Oleic acid Olive oil Polyoxyl 35 castor oil Glycerin Soy fatty acid Peanut oil Polyoxyl 40 hydrogenated castor oil PEG 300 d-α-tocopherol (vitamin E) Rapeseed oil PEG 400 caprylic/capric glycerides PEG 400 Corn oil mono-ditriglycerides Sesame oil Polysorbate 20 Propylene glycol Medium chain (C8/C10) monoand di-glycerides Soybean oil Polysorbate 80 Propylene glycol esters of fatty acids Hydrogenated soybean oil d-α-tocopheryl polyethylene glycol succinate (TPGS) Caprylic/Capric triglycerides derived from coconut oil or palm seed oil Cottonseed oil Sorbitan monolaurate Recently, design of experiments and response surface modeling has been utilized to optimize the screening process [62-64]. Apart from reducing the work load this approach also allowed the investigation of the impact of the individual excipient on the dispersion characteristics [64]. Further work is required to elucidate whether the currently used responses (particle size, solubility) employed in these models are in fact suitable for the optimization of LbDDS, considering that these delivery systems are susceptible to digestion inevitably changing the initial properties of the formulation. III. In vitro characterization of lipid-based drug delivery systems In vitro lipolysis is an important technique employed to mimic the digestion of food and formulation derived lipids such as those contained in LbDDS [65]. Although the protocols for in vitro lipolysis differ in some details between research groups, in vitro lipolysis is commonly carried out in a temperature controlled vessel containing lipolysis medium where the composition of the lipolysis medium is chosen to reflect the conditions (e.g. pH, osmolality, bile salt and phospholipid concentration) of the small intestine during either the fasted or fed state. It should be noted that the physiological values between individuals differ considerably requiring some compromise for the experimental procedure. The pH of the constantly stirred lipolysis medium is measured by a pH-electrode and can be adjusted using a computer-controlled pH-stat device. Lipid digestion commences following the dispersion of the LbDDS and the addition of pancreatic lipase and co-lipase to the lipolysis medium resulting in the release of free fatty acids that are titrated with sodium hydroxide to maintain a constant pH (Figure 2). The consumption of sodium hydroxide is an indirect and unspecific method to measure the progress of lipolysis. For example, the pH of the lipolysis medium could also change due to the hydrolysis of impurities in excipients and the absorption of carbon dioxide by the medium. To account for this “background lipolysis” it is common practice to subtract the sodium hydroxide volume required for the lipolysis of 377 23 Recent developments in oral lipid-based drug delivery N. Thomas, T. Rades, A. Müllertz J. DRUG DEL. SCI. TECH., 23 (4) 375-382 2013 Figure 3 - Schematic of the typical appearance of the lipolysis medium after in vitro lipolysis and ultracentrifugation. An oil phase separates from the aqueous phase in the case of an oil-rich formulation following incomplete lipolysis. Drugs can distribute between the oil and water phase, or precipitate along with calcium soaps. Adapted from [66]. Figure 2 - Illustration of a typical lipolysis set up. The temperaturecontrolled reaction vessel (I) contains the lipolysis medium agitated by a magnetic stirrer (II). The temperature-sensitive pH-electrode (III) is connected to the pH-stat unit (IV) controlling the dispensing of sodium chloride (V) and calcium chloride (VI). The water bath (VII) maintains the temperature of the lipolysis medium at 37°C. Adapted from [66] . that the highest bioavailability resulted after oral administration of SNEDDS in which cinnarizine had the lowest solubility (equivalent to a higher saturation level of 88 % in the pre-concentrate) compared to a SNEDDS with higher solubility (corresponding to 47 % drug saturation in the pre-concentrate). Compared to conventional SNEDDS (drug load well below saturation concentration) similar or increased bioavailability (Figure 4) was also observed for supersaturated SNEDDS (super-SNEDDS) in which simvastatin and halofantrine were present above the equilibrium solubility in the pre- concentrate [66, 76]. It is interesting to note, that cinnarizine, simvastatin, and halofantrine precipitated in an amorphous form during in vitro lipolysis, as evidenced by XPRD [76-78]. In line with the higher energy associated formulation-free lipolysis medium from the sodium hydroxide volume needed for the actual lipolysis experiment. For further chemical characterization of specific lipid digestion products high performance thin layer chromatography and evaporative light scattering have been applied successfully [67, 68]. In addition to the aforementioned chemical characterization of the lipolysis products several physical approaches have been used to assess the evolution of the colloidal phases during in vitro lipolysis. Using cryo transmission electron microscopy Fatouros et al. observed the presence of oil droplets and micelles as the prevailing structures before in vitro lipolysis of SNEDDS was initiated [69]. Over the course of lipid digestion the oil droplets gradually disappeared, giving rise to unilamellar and multilamellar vesicles. These findings were confirmed in subsequent studies using bench top and synchroton small angle x-ray scattering (SAXS) [55, 65, 69, 70]. In the dynamic in vitro lipolysis model employed in Copenhagen the constant addition of calcium chloride controls the rate and extend of in vitro lipolysis by the removal of the digestion products from the lipid surface that would otherwise inhibit further lipolysis [71, 72]. Proceeding lipolytic activity in samples of the medium is inhibited by the addition of lipase inhibitors such as 4-bromobenzene boronic acid. This is followed by the quantification of the drug in the aqueous phase and the pellet obtained after an ultracentrifugation step (Figure 3). LbDDS leading to drug precipitation during dispersion or in vitro lipolysis have been generally regarded unsuitable for effective drug delivery [73, 74]. This paradigm is based on the assumption that only the solubilized drug present in the aqueous phase is available for absorption. Consequently, the general objective of most of the LbDDS development has been to avoid or retard drug precipitation due to concerns of re-introducing a dissolution step of solid drug. A growing body of evidence, however, suggests revision of this paradigm. Several studies have shown that drug precipitation does not necessarily correlate with reduced bioavailability [61, 75-77]. In a recent study investigating the effect of different physicochemical properties of four different SNEDDS on the in vivo performance, Larsen et al. observed comparable areas under the plasma curves of cinnarizine although substantial drug precipitation during in vitro lipolysis was evident for one of the SNEDDS [61]. Moreover, the authors found Figure 4 - Semi-logarithmic plot of the mean plasma concentrations of simvastatin (SIM) administered orally as (l) one capsule of conventional SNEDDS (75 % drug load, corresponding to 67.7 mg SIM), (s) two capsules of conventional SNEDDS (75 % drug load, 135.5 mg SIM), and (u) one capsule of supersaturated SNEDDS (super-SNEDDS, 150 % drug load, 135.5 mg SIM) to six fasted beagle dogs (mean ± SD). AUC0-inf and bioavailability of simvastatin following administration of super-SNEDDS were significantly greater compared to those obtained after administration of dose-equivalent two capsules of conventional SNEDDS. Adapted from [76]. 378 24 Recent developments in oral lipid-based drug delivery N. Thomas, T. Rades, A. Müllertz J. DRUG DEL. SCI. TECH., 23 (4) 375-382 2013 with the amorphous form, the dissolution rate of these compounds was substantially increased compared to the corresponding crystalline forms. The data clearly demonstrate that caution is advised when attempting to predict in vivo results from in vitro performance. Further studies are underway to investigate how the solid state of a compound, drug loading in the pre-concentrates and the ability to supersaturate SNEDDS are linked to their performance in vitro and in vivo. It is perhaps the lack of an absorption step in the currently employed in vitro lipolysis model that makes the in vivo prediction based on in vitro results challenging. Cell culture studies using Caco-2 cells, commonly employed in permeability studies of conventional formulations, have proven difficult in light of the poor tolerance of these cells towards the surfactants and bile salts frequently used in the formulation and characterization of LbDDS [33, 79]. The shortcomings of the Caco-2 cell model might be overcome in the future by the development of mucus producing Caco-2/HT29 co-cultures or Calu-3 cultures that could protect the cell cultures to some degree during permeability studies [80-82]. IV. In vivo testing of LbDDS The number of in vivo studies comparing LbDDS both in vitro and in vivo is still relatively small which is complicated further by the choice of different animal models. Table III provides an overview of recent pharmacokinetic studies concerned with the in vivo performance of a range of LbDDS. Several animal models have been used in these studies including rats, rabbits, minipigs, and dogs. Generally, the selection of the animal model is defined by the research question and economic considerations. Small animals such as rats and rabbits are Table III - Recent in vivo studies with lipid-based delivery systems carried out in different animal species. Type and composition of LbDDS* Drug Major outcome of the study Animal species Ref. SMEDDS: Capryol 90 37 %, Cremophor EL 28 %, Carbitol 28 %, 7 % simvastatin Simvastatin Relative bioavailability SMEDDS (1.5-fold) > commercial tablet Beagle dog [88] MCT (Viscoleo) and LCT (sesame oil) MC-SMEDDS: MCT 25 %, Cremophor RH 40 45 %, mixed MC-glycerides 27 % LC-SMEDDS: LCT 25 %, Cremophor RH 40 45 %, mixed LC-glycerides 27 % Seocalcitol Absolute bioavailability MCT = LCT > MCSNEDDS = LC-SNEDDS Rat [6, 92] MCT (Viscoleo) and LCT (sesame oil) MC-SMEDDS: MCT 25 %, Cremophor RH 40 45 %, mixed MC-glycerides 27 % LC-SMEDDS: LCT 25 %, Cremophor RH 40 45 %, mixed LC-glycerides 27 % Seocalcitol Absolute bioavailability: MC-SNEDDS = LC-SNEDDS > MCT = LCT Minipig [89] Danazol Absolute bioavailability: Solution / suspension in Labrafil M2125CS (9-fold) > aqueous suspension Rat [35] Oil solution: sesame oil, Maisine (1:1) 109.3 mg/kg, 12.1 mg/kg Surfactant solution: Cremophor RH 40 109.3 mg/kg, ethanol 12.1 mg/kg SEDDS: sesame oil, Maisine (1:1) 72.8 mg/kg, Cremophor RH 40 36.4 mg/kg, ethanol 12.8 mg/kg SNEDDS: sesame oil, Maisine (1:1) 72.8 mg/kg, Cremophor RH 40 36.4 mg/kg, ethanol 12.1 mg/kg Probucol Absolute bioavailability: Fasted: SNEDDS ≥ SEDDS ≥ surfactant solution > oil solution > powder Fed: SNEDDS = SEDDS, no food effect, Reduced bioavailability for other formulations with considerable food effect Minipig [38] CrEL-SEDDS: soybean oil, Maisine (1:1) 37.5 %, Cremophor EL 55 %, ethanol 7.5 % CrRH 40-SEDDS: soybean oil, Maisine (1:1) 37.5 %, Cremophor RH 40 55 %, ethanol 7.5 % Danazol Relative bioavailability: CrRH 40-SEDDS > CrEL-SEDDS Beagle dog [94] Rabbit [62] Drug solution or suspension in Labrafil M2125CS SMEDDS: Cremophor EL 30 %, Tween 80 15 %, PEG 400 45 %, Capmul PG-8 10 % Albendazol Relative bioavailability: SMEDDS (1.6-fold) > commercial suspension LC-SNEDDS/LC-super-SNEDDS: soybean oil, Maisine (1:1) 55 %, 35 % Cremophor RH 40, ethanol 10 % MC-SNEDDS/MC-super-SNEDDS: Captex 300, Capmul MCM (1:2) 55 %, 35 % Cremophor RH 40, ethanol 10 % Halofantrine Absolute bioavailability: super-SNEDDS ≥ SNEDDS Beagle dog [77] MC-SNEDDS/MC-super-SNEDDS: Captex 300, Capmul MCM (1:2) 55 %, 35 % Cremophor RH 40, ethanol 10 % Simvastatin Relative bioavailability: super-SNEDDS (1.8-fold) > SNEDDS Beagle dog [76] SNEDDS I: sesame oil 26.3 %, oleic acid 17.6 %, Cremophor RH 40 43.9 %, ethanol 9.7 % SNEDDS IV: sesame oil 5.0 %, oleic acid 3.8 %, Cremophor RH 40 52.7 %, Brij 97 17.6 %, PEG 400 8.8 %, ethanol 9.7 % Cinnarizine Relative bioavailability: SNEDDS IV > SNEDDS I Labrador dog [61] *Terminology as used by the respective authors, CrEL: Cremophor EL, CrRH40: Cremophor RH 40, MCT: medium chain triglycerides, LCT: long-chain triglycerides, SNEDDS: self-nanoemulsifying drug delivery system, SMEDDS: self-microemulsifying drug delivery system, MC-SNEDDS/SMEDDS: formulations containing medium-chain lipids, LC-SNEDDS/SMEDDS: formulations containing long chain lipids, super-SNEDDS: supersaturated SNEDDS. 379 25 Recent developments in oral lipid-based drug delivery N. Thomas, T. Rades, A. Müllertz J. DRUG DEL. SCI. TECH., 23 (4) 375-382 2013 relatively easy to manage and have been commonly employed as in vivo models for absorption studies from drug solutions and powders [83]. On the other hand, larger animals are more suitable to study the bioavailability from formulations [83]. With regard to the in vivo assessment of LbDDS, the most frequently used species are the rat [35, 84-86] and the dog [53, 87, 88] whereas only very limited data is available for minipigs [38, 89, 90]. While rats are less resource-intensive than other animals they might not be the ideal species to investigate LbDDS as they do not store bile in a gall bladder, but instead, have a continuous bile flow, possibly affecting the digestion process outlined above [83, 89]. In addition, the small size of rats limits the maximum amount of formulation that can be administered [83, 91]. This can have consequences for the dynamics of dispersing formulations as in the case of SEDDS and SNEDDS [89]. Following the administration of simple LbDDS composed of medium-chain (MC) and long chain (LC) triglycerides the bioavailability of the drug seocalcitol did not differ when evaluated in rats [92]. The obtained in vivo data were in line with solubility studies carried out in simulated intestinal media containing lipolysis products of the employed LbDDS. Compared to MCT and LCT a slight decrease in the bioavailability was observed when rats were given either MC- or LC-SNEDDS [6]. However, when studied in minipigs, the MC-SNEDDS caused an increased bioavailability compared to the treatment with MCT. The authors hypothesized that the limited volume of intestinal fluids present in the rat might not generate a finely dispersed nanoemulsion but “viscous gel-like structures” that could not unfold its full potential [89]. This example emphasizes the need for the appropriate selection of the animal model when evaluating LbDDS. According to the previous discussion there might be some merit in the use of larger animals to study LbDDS. Each animal model requires careful consideration, or at least awareness, for its inherent drawbacks. Critical factors to consider include the anatomy, pH, gastrointestinal motility, and surface area of the intestine [90, 93]. While minipigs resemble the gastrointestinal physiology in humans closer than dogs [83, 90] their intestinal anatomy and length differ in some aspects [83]. The greater experience with dog models along with the good agreement with many human data in previous bioavailability and food effect studies renders the dog a suitable animal model for the assessment of LbDDS. Until comparative studies across several species are available (which would be very desirable) it appears that both dogs and minipigs are better suitable for the assessment of LbDDS than rats. Ultimately, a larger set of in vivo data will help elucidate a more refined picture of the full potential of LbDDS. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. * 18. Lipid-based drug delivery has great potential for the delivery of poorly water-soluble, lipophilic drugs. However, to fully realize this potential our knowledge about these complex delivery systems has to advance considerably. The current review sought to highlight some of the challenges encountered in the development and characterization of LbDDS. Insights into drug absorption from LbDDS on a molecular level will aid in their rational formulation design. 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