Solubility Enhancing Excipients Functional Effects on Drug Transporters and Metabolizing Enzymes: In Vitro Folklore or In Vivo Reality?

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1 Solubility Enhancing Excipients Functional Effects on Drug Transporters and Metabolizing Enzymes: In Vitro Folklore or In Vivo Reality? Gregory Knipp, Ph.D. Associate Professor AAPS Annual Meeting 2014 November 5, 2014

2 Objectives Introduction Highlight potential mechanisms by which excipient classes have been associated with transporter or enzyme interactions Highlight the strengths and limitations of in vitro assays Discuss representative in vitro data Highlight some of the important physiological considerations for in vivo performance Discuss representative in vivo cases Conclude with a highlight of future needs.

3 Solubility vs. Direct Effect: Considerations There are two main issues to consider: Do excipient induced in vitro/in vivo solubility increases correspond to saturation of Michaelis Menten Kinetics for transporters or enzymes? Rarely do we see a good estimate of the increase in solubility correlated with excipient effects P gp saturation has been debated in the field Do correlations made in vitro hold in vivo? Do the excipients directly act on the transporters or enzymes: Several mechanisms have been proposed in the literature for a direct effect Strength of the evidence?

4 Some Facts Excipients are not inert! US warning on HIV drug excipient A warning has been issued over the use of the oral solution formulation of Glaxo Welcome's protease inhibitor amprenavir (Agenerase). The company has written to doctors to draw attention to potential safety concerns relating to the large amount of propylene glycol in solution, which was included in the formulation to achieve adequate solubility of amprenavir.it is now recommending that the solution should be used only when Agenerase capsules or other protease inhibitor formulations are not therapeutic options. The Pharmaceutical Journal May 2000, 264 (7095), 685 Amprenavir is believed to be permeability limited by intestinal CYP3A4 activity (Dufek MB, et al. Drug Metab Dispos. 41: , 2013), thus enzyme saturation could be an effect?

5 Propylene Glycol Largely considered as a solubility enhancer, with a negligible effect on P gp for substrates. Appeared to have a slight, but not statistically different effect on small intestinal transit times (SITT) that may be linked to absorption in humans. There was no effect on SITT in dogs. Hypertonic effect is larger than most co solvents. PEG 400 does increase small intestinal transit times. Commonly used as a co solvent with other P gp inhibitors. J.D. Schulze, et al. Impact of formulation excipients on human intestinal transit. J Pharm Pharmacol. 58: (2006).; J.D. Schulze, et al. Excipient effects on gastrointestinal transit and drug absorption in beagle dogs. Int J Pharm. 300:67 75 (2005).

6 Efflux Pump Inhibitors Proposed Polymer Interaction Mechanisms M. Werle. 2008, Pharm Res. 25:

7 Proposed Qualified Polymer Pgp Interactions Proposed mechanisms (figure on the next slide, Panels listed below): A. The polymer traverses the membrane and act intracellularly to deplete ATP. B. The polymer may interact with the membrane and block the function of the transporter. Mechanistic explanations vary, although many believe that it arises from membrane fluidization. C. Polymer drug conjugates may traverse the membrane and are not effected by the efflux pump. D. There is the potential that the polymer can traverse the membrane and directly interact with the ATP binding sites. E. The final illustration suggests that the polymer will directly interact with the pump and inhibit it s function. M. Werle. Natural and Synthetic Polymers as Inhibitors of Drug Efflux Pumps. Pharm Res. 25: (2008).

8 Proposed Mechanisms for Surfactants Most believe that the mechanism is membrane disruption or fluidization to alter P gp and other ABC transporters like BCRP. TPGS is thought to rigidize membranes Cremophor EL and Tween 80 fluidize Off target effects can occur, e.g. drug targets or other transporters being effected (e.g. MRP2) Lipid membranes differ in phospholipid composition, so cell types may behave differently. G. Cornaire, J. Woodley, et al. Impact of excipients on the absorption of P glycoprotein substrates in vitro and in vivo. Int J Pharm. 278: (2004).

9 In Vitro Models Potential Confounding Variables that can Alter Data Interpretation for Excipient Transporter or Enzyme Interactions

10 API A B C D Routes of Permeability A) Influx transporter mediated B) Passive transcellular C) Passive transcellular and efflux D) Passive paracellular E) Metabolism F) Efflux of the metabolite(s) There can exist a number of combinations of parallel routes of permeation that can cocontribute to the net permeation of an API during the absorption process. E? F? Met. G. Zografi, G. Knipp, A. Newman. Commentary: Assessing the Performance of Amorphous Solid Dispersions. J Pharm Sci. 101: (2012) 10

11 Variability in Cell Based Assays The FDA is now paying significant attention to cell based permeability assay variation. Affinity for multiple transporters can obfuscate physiologically relevant predictions Inherent variability leads to differences in P APP values for the same drug Divided into three categories 1. Cell Culture Methods/Techniques 2. Transport Experiments 3. Data Analysis FDA and DIA Critical Path Transporter Workshop, North Bethesda, MD, Oct. 2 3, 2008 Also see: Donna Volpe publications on cell line variability.

12 Caco 2 (other) Cell Limitations They do have variable degrees of transporter and enzyme expression: CYP3A4/5 poorly expressed metabolites are effluxed by P gp and others. P gp seems to be highly expressed. They lack a glycocalyx and mucus layer that mimics in vivo. Highly passaged and can be improperly numbered Media variation influences transporter expression Permeability conducted in buffers, not physiologically relevant media

13 A. Avdeef. Chapter 2. Transport Model. Absorption and Drug Development: Solubility, Permeability, and Charge State. John Wiley & Sons, Hoboken, NJ, 2003, pg. 14.

14 Tonicity Osmolarity is the number of osmoles of a solute present in 1 L of solution, and is influenced by charge. Osmolality accounts for mass density of the solution, the media density may change from polymers Tonicity is defined by the osmolality in the extracellular compartment in comparison to the osmolality in the intracellular cytoplasm. Permeability assays are often conducted in media that is isotonic with the cytoplasm. Hypertonic extracellular solutions can cause cell shrinkage Hypotonic extracellular solutions can cause cell swelling and membrane damage Based on my knowledge, most excipient studies revealing transporter/enzyme effects do not highlight tonicity. Tonicity may affect predominant route of permeation.

15 Hyperosmosis and Paracellular Permeability in Caco 2 Cells. Increasing osmotic pressure reduces TEER values, increases the paracellular marker permeation and alters permeability of substrates. Care needs to be taken when working with higher concentrations of polymers in Caco 2 cells. H. Inokuchi, et al. The effect of hyperosmosis on paracellular permeability in Caco 2 cell monolayers. Biosci Biotechnol Biochem. 73: (2009).

Polyamidoamine (PAMAM) Polymers Anionic and cationic PAMAMs appear to more dramatically disrupt the tight junctions in Caco 2 cells. PAMAMs can bind to the lipid bilayer in in vitro Caco 2 cells.

16 Polyamidoamine (PAMAM) Polymers Anionic and cationic PAMAMs appear to more dramatically disrupt the tight junctions in Caco 2 cells. PAMAMs can bind to the lipid bilayer in in vitro Caco 2 cells. Questions: 1) Is isotonicity maintained in culture systems? 2) How would the glycocalyx and mucus influence binding in vivo relevance? K.M. Kitchens, R.B. Kolhatkar, P.W. Swaan, N.D. Eddington, H. Ghandehari. Transport of Poly(Amidoamine) Dendrimers across Caco 2 Cell Monolayers: Influence of Size, Charge and Fluorescent Labeling. Pharm Res. 23: (2006).

17 PAMAM Dendrimer Conjugates and Pgp Propranolol conjugated to PAMAM dendrimers increased solubility and blocked Pgp?, increasing Caco 2 permeability Propranolol conjugated to lauroyl PAMAM dendrimers also had a similar effect with reduced dendrimer cytotoxicity. Paracellular markers increased with dendrimers Conjugates proposed to be endocytosed, however Caco 2 cells have very low endocytosis. A. D Emanuele, R. Jevprasesphant, J. Penny, D. Attwood. The use of a dendrimer propranolol prodrug to bypass efflux transporters and enhance oral bioavailability. J Contr Rel. 95: (2004).

18 In Vivo Models Providing a Physiologically Relevant Environment for the Interpretation of Potential Excipient Transporter or Enzyme Interactions

Disintegration/ Deaggregation Dissolution

Solubility Solubility Enhanced API Free API

Bile Potential confounders: food, ph, protein

Commentary: Assessing the Performance of

19 Disintegration/ Deaggregation Dissolution Luminal Absorption Crystalline Form Low Solubility Solubility Enhanced API Free API Particle Intestinal flora Mucus/ Glycocalyx Bile Potential confounders: food, ph, protein binding, etc. G. Zografi, G. Knipp, A. Newman. Commentary: Assessing the Performance of Amorphous Solid Dispersions. J Pharm Sci. 101: (2012) Absorption

20 PEG Affects In Situ P gp/efflux PEG does impact the Efflux Ratio of Rhodamine 123, but not Lucifer Yellow, suggesting PEG is affecting Pgp mediated efflux. Q. Shen, et al. Modulation of intestinal P glycoprotein function by polyethylene glycols and their derivatives by in vitro transport and in situ absorption studies. Int J Pharm. 313:49 56 (2006).

21 Surfactants and Efflux Improving Oral Bioavailability by Reducing Efflux Liability (Borgman et al., Clinical Pharmacology & Therapeutics (2005) 77, 24 32) Drug Drug The Efflux Ratio (ER) is determined by dividing the (Basolateral to Apical)/(Apical to Basolateral) permeability coefficients in in vitro models. When the ER > 3, it is presumed that efflux transport is controlling absorption across the barrier. Here, talinolol in buffer is approximately 10. TPGS enhanced the Apical to Basolateral permeability and decreased the Basolateral to Apical permeability compared to talinolol alone or in the Polaxamer 188 formulation, suggesting an inhibitory effect of TPGS on P gp across the Caco 2 cells..

22 Surfactants and Efflux Improving Oral Bioavailability by Reducing Efflux Liability (Borgman et al., Clinical Pharmacology & Therapeutics (2005) 77, 24 32) In the case of Talinolol, the TPGS also enhanced the bioavailability compared to talinolol alone or in the Polaxamer 188 formulation, suggesting a clinical inhibitory effect of TPGS on P gp in the intestine and that the Caco 2 model was a good surrogate. Solubility effect?

23 Breast Cancer Resistance Protein Formulation Strategy Effects BCRP only below the critical micelle concentration, thus the monomer is believed to be driving the effect. Above the CMC, the hypothesis could be that the micelle may compete for drug distribution, which has been suggested for a number of surfactants that influence absorption.

Nonionic Surfactants on CYP3A4 Nonionic surfactants had a concentration dependent effect on CYP3A4 metabolism of midazolam consistent with mixed competitive inhibitory model mechanism.

24 Nonionic Surfactants on CYP3A4 Nonionic surfactants had a concentration dependent effect on CYP3A4 metabolism of midazolam consistent with mixed competitive inhibitory model mechanism. The reduction in the 1 Hydroxylmidazolam indicates excipient inhibition of CYP3A4 based on the metabolite PK in rats. Due to CYP diversity and the potential issues based on species differences, screening and extrapolation need to be considered carefully. KTZ is ketoconazole, a known CYP3A4 inhibitor: polysorbate 20 (Tween 20); polyoxyl 35 castor oil (EL35); polyoxyl 40 stearate (S40); and poloxamer 188 (F68). Ren X, Mao X, et al. Eur J Pharm Sci. 36: (2009).

25 Future Directions Develop standardized testing protocols for in vitro and in vivo assays examining excipient transporter effects. Improve the translatability of in vitro results to better predict in vivo effects. Determine the mechanisms behind excipient mediated drug transporter or enzyme effects. Develop rational screening trees for the excipient selection. Develop better safety testing for solubility enabling excipients. Suggestions?

26 Acknowledgements Dr. Wyatt Roth Dr. Stephen Carl Dr. David Lindley Dr. Olafur Gudmundsson Dr. Yan Xu Dr. Qing Wang Dr. Jonathan Goole Dr. Lori Karpes Mr. Chris Kulczar Ms. Monika Lavan Mr. Aimable Ngendahimana Ms. Kelsey Lubin Dr. Dea Herrera Ruiz Dr. Stephanie Mowery Dr. Li Fang Pan Funding and Research Support Purdue Translational Pharmacology CTSI Core Dane O. Kildsig Center for Pharmaceutical Processing Research Department of Industrial and Physical Pharmacy American Association of Pharmaceutical Scientists

27 Nutrient and Xenobiotic Transporters Solute Carrier (SLC) 43 Subfamilies > 300 members identified Generally influx or secretory efflux transporters PepT1, OATs, OATPs ATP Binding Cassette (ABC) 7 Subfamilies 50 members presently identified Generally effluxmultidrug resistant transporters P glycoprotein, MRPs Nomenclature: Transporter Type; Family Number; Species Variant. Example: Human PepT1 SLC15A11

28 Passive Paracellular Permeation Hydrophilicity. Molecular Size and Shape pka of the ionizable groups Linear increase in permeability with increasing concentration Adjuvants can open tight junctions and increase transport Facilitative/Active Transcellular Permeation Affinity (Km), Capacity (Vmax/Jmax) Concentration dependent saturation Expression level (constitutive, induced). Function (drug drug & drug nutrient interactions, competitive inhibition.) Excipients like surfactants can limit the effects of efflux by Pgp or BCRP. P Barrier = P Para + P Passive, Trans + P ±... Active, Trans 1 + P Active n, Trans Passive Transcellular Permeation Lipophilicity Hydrogen Bonding Potential Hydrophobicity Molecular Size and Shape pka of the ionizable groups Linear increase in permeability with increasing concentration Dissolution/Solubility limited with high lipophilicity Efflux and metabolism can affect the net absorption of drug G. Zografi, G. Knipp, A. Newman. Commentary: Assessing the Performance of Amorphous Solid Dispersions. J Pharm Sci. 101: (2012)

29 Cell Based Assays Permeability Coefficient ΔQ/Δ t : amount of compound appearing on the receiver side as a function of time P app = Δ Q / Δ t A C o 60 1 (cm sec) 1 = 1 A : surface area of the filter support C 0 P app P M P ABL / F / + : initial concentration of compound applied to the donor side 1 C

30 The Link Between PERMEABILITY and SOLUBILITY FICK S FIRST LAW: FLUX, not permeability, is solubility dependent! FLUX only relates to passive permeability. Permeability is not uniform when a substrate has affinity and capacity driven transport and metabolism These are saturable processes, thus permeability below saturation is concentration dependent.

One of the major factors affecting oral drug bioavailability is the activity of intestinal transport proteins, particularly for

One of the major factors affecting oral drug bioavailability is the activity of intestinal transport proteins, particularly for REVIEW ARTICLE Influence of excipients on drug absorption via modulation of intestinal transporters activity P. Thakkar Hetal, L. Desai Jagruti Department of Pharmacy, Faculty of Technology and Engineering,