Article pubs.acs.org/molecularpharmaceutics
Leveraging BCS in Development: A Case Study Cherokee Sue Hoaglund Hyzer,† Hala M. Fadda,‡ Jole O. Rodriguez,† and Aktham Aburub*,† †
Small Molecule Design and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, United States ‡ Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Butler University, Indianapolis, Indiana 46208, United States ABSTRACT: In this work, we discuss leveraging the Biopharmaceutics Classification System (BCS) in the development of edivoxetine HCl, a selective norepinephrine reuptake inhibitor. First, the biopharmaceutical and in vivo data are presented and discussed. Solubility studies indicate that edivoxetine HCl meets the BCS “highly soluble” criteria. To determine permeability classifications, in vitro intestinal Caco2 epithelial cell model with and without cyclosporin A (CsA), a common P-glycoprotein (P-gp) inhibitor, were conducted. Pharmacokinetic (PK) data obtained across phase 1 and 2 clinical studies where single and multiple doses range from the lowest to the highest strength are presented. Neither the Caco-2 nor the in vivo data on their own were sufficient to conclusively classify edivoxetine as highly permeable. However, collectively the data were utilized to support high permeability and consequently BCS1 classification of edivoxetine HCl. BCS1 classification was leveraged throughout development to assess the risk associated with not conducting relative bioavailability (RBA) studies and avoiding bioequivalence (BE) studies. Examples are presented where formulation changes were made between phase I (drug in capsule/drug in bottle formulations) and phase II (tablet) trials in addition to phase III (tablet) and commercial (smaller tablet) without having to conduct any in vivo comparability studies. For the first change, BCS was leveraged to avoid conducting a RBA study even before obtaining official BCS classification. For the later change, official BCS1 classification was relied upon to avoid conducting a BE study. KEYWORDS: edivoxetine HCl, BCS, solubility, permeability, biowaiver
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INTRODUCTION As a new molecular entity (NME) progresses through the various phases of clinical development, it is fairly common for the drug substance and the drug product attributes to undergo multiple changes. Such changes are aimed at optimizing the drug substance or drug product manufacturing processes and/ or the formulation. Specifically, changes to the drug product are almost inevitable in particular in the fast proof of concept (POC) approach, in which simple and often noncommercializable formulations like a drug in capsule or drug in bottle are utilized early in development.1,2 Such formulations allow for rapid and cheap development cost (minimize the at risk investment) while enabling phase I/II clinical trials. Changes to the drug substance physical properties (e.g., particle size, solid form)3−7 or formulation (e.g., unit formula, manufacturing platform)8−11 may have an impact on the rate and/or extent of absorption (bioavailability) and, hence, the clinical outcome (safety, tolerability, and efficacy). In order to understand the impact of drug substance and/or drug product changes on bioavailability, relative bioavailability (RBA) or bioequivalence (BE) studies are conducted.12 RBA studies are typically used prior to starting pivotal clinical studies and are utilized to mitigate a business risk (i.e., taking the wrong dose into the next clinical study). BE studies are typically conducted post pivotal to satisfy a regulatory requirement. Such in vivo studies © 2015 American Chemical Society
(RBA/BE) are costly, time-consuming, and depending on the power are susceptible to false negative/positive results. The Biopharmaceutical Classification System (BCS) is a scientific approach for classifying drug substances and products based on the drug substances’ aqueous solubility and intestinal permeability and the drug products’ in vitro multimedia dissolution.13 A drug substance needs to be classified as BCS1 to qualify for a BCS based biowaiver.14,15 Biowaiver extension to other classes, specifically BCS3, has also been discussed16−19 and recently proposed in a FDA draft guidance.20 Edivoxetine HCl (Figure 1) is a selective norepinephrine reuptake inhibitor that has been investigated for the treatment of major depressive disorders (MDD), monotherapy or adjunct therapy. Edivoxetine HCl has a pKa of 8.4 and Log P of 1.4 and is not considered a narrow therapeutic index drug. BCS classification of edivoxetine HCl is described in this work. The biopharmaceutical and in vivo data are presented and discussed. Solubility studies indicate that edivoxetine HCl meets the BCS “highly soluble” criteria. To determine the Received: Revised: Accepted: Published: 3685
June 7, 2015 July 24, 2015 September 13, 2015 September 13, 2015 DOI: 10.1021/acs.molpharmaceut.5b00450 Mol. Pharmaceutics 2015, 12, 3685−3690
Article
Molecular Pharmaceutics
cin. For preparation of Transwell assay plates, cells were harvested from 175 cm2 T150 flasks using a solution of 0.25% trypsin and 2.21 mM EDTA (Gibco Life Technologies), and cells were seeded on 12-well Costar Transwell plates containing collagen-coated, microporous, polycarbonate filter membranes. Caco-2 monolayers were grown to confluence on the filter membranes in a humidified atmosphere containing 5% CO2 at 37 °C. Integrity of the cell layers was confirmed by measuring transepithelial electrical resistance (TEER) and Papp values of selected reference compounds (e.g., Lucifer yellow, pindolol, and atenolol). Permeability Methods. Dosing solutions of edivoxetine HCl were prepared in the pH 7.4 permeability assay buffer (HBSS) at concentrations of 1.77, 17.7, and 177 μM. Internal permeability standards, pindolol and atenolol, were included in each dosing solution. Pindolol was at a concentration of 10 μM, and atenolol was at a concentration of 100 μM. Permeability experiments were conducted in absorptive and secretory directions in quadruplicate (n = 4) at each concentration. For the apical-to-basolateral (A-to-B) permeability, dosing solutions were applied to the apical side, and for the basolateral-to-apical (B-to-A) permeability, dosing solutions were applied to the basolateral side. The receiver was a drugfree buffer at pH 7.4. During the permeability period, cell monolayers were incubated at 37 °C with 5% CO2 and 90% relative humidity. The plates containing the cell monolayers were shaken using a Lab-line Instruments Titer Plate Shake. The shaker was placed inside the incubator. The sampling times were 15, 30, 60, and 90 min for the receivers, and 90 min for the donors. The sample volume from the receiver was replaced with fresh drug-free buffer after each sample collection. In the case in which cells were dosed with CsA, a 30 min preincubation step was included with buffer containing 10 μM CsA. All samples were assayed by LC−MS/MS using electrospray ionization. A Sciex API 3000 LC−MS/MS mass spectrometer was used in multiple reaction monitoring (MRM) mode (source: TurboIonSpray, nebulizing gas 8, curtain gas 10, CAD gas 6, ISV 4500v, temperature 350 °C). The LC conditions consisted of Thermo BDS Hypersil C18, 3 μm 2.1 × 30 mm column. The mobile phase comprised mobile phase A (90% water and 10% ammonium formate buffer (40 mM NH4OH to pH 3.5 with 88% formic acid)) and mobile phase B (90% acetonitrile and 10% ammonium formate buffer (40 mM NH4OH to pH 3.5 with 88% formic acid)) with a flow rate of 0.3 mL/min. Gradient: 100% A to 50% B at 0.5 min to 100% B at 1 min and 100% B at 2.5 min to 100% A at 2.6 min continuing to 4 min. The apparent permeability, Papp, and percent recovery were calculated as follows:
Figure 1. Edivoxetine HCl.
permeability classification, in vitro studies conducted using the intestinal Caco-2 epithelial cell model and pharmacokinetic (PK) data obtained across phase 1 and 2 clinical studies were utilized. While neither in vitro nor in vivo data on their own were sufficient to conclusively classify edivoxetine as highly permeable, collectively the data were utilized to support high permeability classification and subsequently gain agreement from FDA to classify edivoxetine HCl as BCS1. After discussing in vitro as well as in vivo data supporting BCS1 classification, leveraging BCS1 classification throughout development to assess the risk associated with not conducting RBA studies and avoiding BE studies is discussed. Examples are presented where formulation changes were made between phase I (drug in capsule/drug in bottle formulations) and phase II (tablet) trials in addition to phase III (tablet) and commercial (smaller tablet) without having to conduct any in vivo comparability studies. For the first change, preliminary BCS was leveraged to avoid conducting a RBA study before obtaining official BCS classification. For the later change, official BCS1 classification was relied upon to avoid conducting a bioequivalence (BE) study.
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EXPERIMENTAL SECTION Materials. Edovoxetine HCl was obtained from Eli Lilly and Company (Indianapolis, IN). Pindolol, atenolol, and cyclosporin A (CsA) were purchased from Sigma-Aldrich (St. Louis, MO). Hanks’ balanced salt solution (HBSS) and HEPES (N[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) were purchased from Gibco Life Technologies (Grand Island, NY). D-Glucose was purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical or high-performance liquid chromatography (HPLC) grade. Methods. Solubility. Solubility of edivoxetine HCl was determined using the shake-flask method in pH 1.2, 3.0, 4.5, 6.0, and 7.4 (hydrochloric acid, acid phthalate, acetate, phosphate, and phosphate buffers; respectively) all prepared according to USP 28-NF 23 at 37 °C. A target 200 mg/mL drug concentration was placed in test tubes, to which the appropriate medium was added, and the tubes were shaken at a speed of 200 rpm for 48 h. At the end of the experiment, all samples were analyzed using HPLC and the equilibrium pH was recorded. The HPLC system consisted of an Agilent 1100 series with a PDA detector (Agilent Technologies, Santa Clara, CA) and a Waters SymmetryShield, RP8, 5 μm, 4.6 × 250 mm column. The mobile phase comprised 80% mobile phase A (10 g/L ammonium chloride/0.05% hydrochloric acid) and 20% mobile phase B (acetonitrile) with a flow rate of 1.0 mL/min. The PDA detector wavelength was set at 280 nm. Solubility experiments were conducted in triplicate. Permeability. Cell Culture. Caco-2 cells (passages 61, 62) were grown in a maintenance medium composed of Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1 mM sodium pyruvate, 100 μM nonessential amino acids, 4 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomy-
Papp = (dCr /dt )Vr /ACo
(1)
percent recovery (%) = 100(VrCrfinal + VdCdfinal)/VdC0
(2)
where dCr/dt is the slope of the cumulative concentration in the receiver compartment versus time in μM s−1, Vr is the volume of the receiver compartment in cm3, A is the area of the cell monolayer (1.13 cm2 for 12-well Transwell), Vd is the volume of the donor compartment cm3, Cfinal is the cumulative r receiver concentration in μM at the end of the incubation period, Cfinal is the concentration in μM of the donor at the end d of the incubation period, and C0 is the concentration of the dosing solution in μM. 3686
DOI: 10.1021/acs.molpharmaceut.5b00450 Mol. Pharmaceutics 2015, 12, 3685−3690
Article
Molecular Pharmaceutics Stability. Chemical stability data has been generated utilizing USP simulated gastric fluid (SGF) without enzymes pH 1.1 and USP simulated intestinal fluid (SIF) without enzymes pH 6.8. Samples (triplicates) at a concentration of 0.001 mg/mL were incubated at 37 °C in SGF for 3 h and in SIF for 4 h. Initial and final time points were analyzed utilizing a stability indicating HPLC method. The HPLC system consisted of an Agilent 1100 series with a PDA detector (Agilent Technologies, Santa Clara, CA) and a Zorbax Bonus RP, 3.5 μm, 4.6 × 75 mm column. The mobile phase comprised mobile phase A (90% 10 mM pH 5.9 ammonium acetate buffer and 10% acetonitrile) and mobile phase B (5% 10 mM pH 5.9 ammonium acetate buffer and 95% acetonitrile) with a flow rate of 1.0 mL/min. Gradient: 100% mobile phase A to 100% mobile phase B at 38 min; 100% mobile phase B until 40 min after which switching back to 100% A. The PDA detector wavelength was set at 280 nm. Dissolution. In vitro dissolution profiles were generated using USP apparatus I (100 rpm) in media that spanned the physiological pH range (0.1 N HCl, acetate buffer pH 4.5 (USP), and phosphate buffer pH 6.8 (USP)) at 37 °C.
concentrations of edivoxetine HCl chosen for the study were 1.77, 17.7, and 177 μM. Subsequently, the highest proposed dose was increased to 18 mg edivoxetine. Conclusions from the study should not be impacted by the increase in dose since a higher dose should not have an impact on the absolute estimate of permeability. In addition, efflux is less of a factor as the dose is increased. The internal control compounds, pindolol and atenolol with high and low permeability, respectively,14,15 were included in each permeability experiment. A bidirectional permeability assay was performed with and without cyclosporin A (CsA), a common P-glycoprotein (P-gp) inhibitor, to study the efflux of edivoxetine in Caco-2 cells. In the absence of CsA (Tables 1 and 2), absorptive permeability values of edivoxetine HCl approximated that of the high permeability marker, pindolol, and the compound exhibited slight efflux (i.e., efflux ratio 85% release within 15 min. An example can be seen in Figure 4. The dissolution rate tablet-totablet variability RSD was consistently 99%) will be in the ionized 3688
DOI: 10.1021/acs.molpharmaceut.5b00450 Mol. Pharmaceutics 2015, 12, 3685−3690
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Molecular Pharmaceutics
Figure 3. Relationship between dose and steady state AUC0−24 (top panel) and steady state Cmax (bottom panel) following daily administration of edivoxetine.
clinical studies, and tablet 2 are identical from a composition stand point except for film coating level and color. Edivoxetine phase II clinical studies were designed to be potentially pivotal. Hence, a regulatory risk was associated with formulation changes made beyond phase II. To mitigate this risk, all tablets were designed to be rapidly dissolving and an official FDA BCS1 classification was pursued and granted. A smaller tablet for better patient compliance was later developed and was intended for commercialization. Despite the difference between the dosage form that was used in phase III and the intended commercial dosage form, a biowaiver of an in vivo bioequivalence study was secured.
Table 3. Assessment of Dose Proportionality Following Daily Administration of Edivoxetine steady state PK parameter
power model eq
dose range
predicted geometric mean
rdnm (90% CI)a
AUC (ng h/ mL)
20.62 × dose1.10
6
148.38
1.15 (1.11, 1.19)
Cmax (ng/mL)
2.26 × dose1.07
24 6
683.06 15.40
1.10 (1.04, 1.18)
24
67.99
a
CI = confidence interval; rdnm = ratio of dose normalized modelpredicted geometric mean.
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CONCLUSION Utilizing a combination of in vitro and in vivo data, edivoxetine HCl and products are classified as BCS1. This classification,
negatively impact stability. Therefore, it was removed leading to tablet 2. Tablet 1 and tablet 2 were used to support phase II clinical studies. Tablet 3, which was used to support phase III 3689
DOI: 10.1021/acs.molpharmaceut.5b00450 Mol. Pharmaceutics 2015, 12, 3685−3690
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Molecular Pharmaceutics
Figure 4. Edivoxetine HCl tablet dissolution (multimedia; 0.1 N HCl, pH 4.5 acetate buffer and pH 6.8 phosphate buffer) vs time.
preliminary at first and then official, enabled aggressive development using simple formulations for early clinical studies followed by switching to the commercial formulation without having to conduct in vivo bridging studies.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: 317-655-0869. Fax: 317-655-2770. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge and thank Drs William Kielbasa and Debra R Luffer-Atlas for their help and insightful discussions. REFERENCES
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DOI: 10.1021/acs.molpharmaceut.5b00450 Mol. Pharmaceutics 2015, 12, 3685−3690
