Biopharmaceutics Classification System

(BCS)-Based Biowaiver Studies of Lenalidomide Capsules (25mg) – An Alternative To In Vivo Bioequivalence Studies For Generic Oncology Drug Products Alswisi M, Boujbel, et al. Les Laboratoires Medis, Nabeul, Tunisia Abstract. Lenalidomide, commercialized as Revlimid®, is an immunomodulatory drug, approuved as a therapy for Multiple Myeloma and deletion 5q Myelodysplastic syndromes. This molecule shows a promising therapeutic potential in other hematologic malignancies. According to the European Medicines Agency (EMA), lenalidomide is considered as a fully absorbed drug substance, but there is no sufficient data on solubility that enables its BCS classification. So, as per the International Council for Harmonization (ICH) guideline, a solubility study have been proceeded to prove that lenalidomide is a highly soluble compound. This demonstration requires the investigation in four buffer solutions within the range of pH 1- 6.8 (including pKa) at 37±1 °C and we have proceeded with a nominal concentration (1.0 mg/ml) which represent ten times the target concentration (0.1 mg/ml). The main reason of conducting this study is to evaluate the possibility of a BCS-based biowaiver for the consent of a generic oncology drug product unescorted by any further in vivo bioequivalence (BE) studies, also, to substantiate that Lenalidomide can be classified as high solublity and high permeablity, i.e., BCS class I. The main objective in establishing the BCS-based biowaiver procedure is to minimize the need for in vivo BE studies, that would reduce the exposure of healthy volunteers to oncology drugs such as lenalidomide, to prove BE. . The test product and Revlimid were evaluated by in vitro methods as stated by the ICH guidelines in three dissolution buffers: pH 1.2, 4.5, and 6.8, also in HCL 0.01N in compliance with the US Food and Drug Administration (FDA) dissolution method, to assess bioequivalency among the two products.                

 Keywords: Lenalidomide; BCS-based biowaiver; Oncology; Bioequivalence; Pharmacokinetics Introduction The required time for obtaining the marketing authorization depends on the development process and clinical trials. Establishing bioequivalence (BE) and bioavailability can cost up to 300.000$, not to mention the long duration to achieve, contrarily to in-vitro tests which are relatively economical (2.000$) and rapid [1]. To evaluate bioequivalency among two pharmaceutical products, the active substance in the generic product must display a similar oral bioavailability as the originator product [2]. Granting a biowaiver has become possible for solid oral immediate release (IR) drugs by means of in-vitro tests based on the Biopharmaceutics Classification System

(BCS) [2]. Amidon has introduced the BCS to classify drug substances upon 3 principal factors, namely, the permeability, the aqueous solubility of the Active Pharmaceutical Ingredient (API), and the dissolution profile of the drug product [2,3]. The BCS is adopted by the US FDA as a regulatory tool to grant biowaivers, which can reduce the exposure of healthy volunteers to drugs under development. In the case of oncology drug products, the BCS-based approach grants numerous advantages when it comes to the complexities involved in conducting the in-vivo BE trials especially for cytotoxic oncology drug products. It is often considered as unethical to carry out BE studies in healthy volunteers using cytotoxic drugs.

Therefore the BCS-based biowaiver presents an alternative to avert conducting BE studies in humans [2].Since BCS-based biowaiver is only granted for highly soluble drug substances, the aim of this study is to justify the merit of lenalidomide as a generic oncology drug product to be a candidate for such registration procedure. General characteristics Lenalidomide is commercialized by Celgene Corporation under the name of Revlimid®. Lenalidomide (3-(4′aminoisoindoline-1′-one)-1-piperidine-2, 6-dione; Fig. 1) [4], is a thalidomide derivative, a second-generation immunomodulatory drug, administrated through oral route. It has anti-inflammatory, anti-angiogenic, antineoplastic and pro-erythropoietic properties [5]. The biochemical mechanism of lenalidomide consists in binding to an E3 ubiquitin ligase complex protein, cereblon, modifying its subsequent effects [6]. This interaction was demonstrated to be related to the antitumor and the immunomodulatory properties of lenalidomide [6]. The empirical formula, the gram molecular weight and other chemical properties of lenalidomide are listed in table 1[7]. Table 1: Chemical proprieties of lenalidomide Molecular formula C13H13N3O3 Molecular weight 259.3 Octanol-water partition coefficient -0.43 pka 2.31 As shown in figure 1, lenalidomide has an asymmetric carbon atom. The optically active forms of lenalidomide are the S- and R-enantiomers produced as a 50:50 racemic mixture [6]. Lenalidomide is commercialized for oral administration as hard gelatin capsules with seven strengths:  2.5, 5, 7.5, 10, 15, 20, and 25 mg [6]. Therapeutic indication Lenalidomide is approved as a therapy for hematologic malignancies. It\’s prescribed in combination with dexamethasone for the treatment of Multiple Myeloma (MM), but as a monotherapy, it\’s approved for the treatment of acute myeloid leukemia (AML), transfusion-dependent anemia due to Myelodisplastic Syndrome (MDS) interrelated with deletion 5q and relapsed or refractory Mantle Cell Lymphoma (MCL) [6]. Figure 1: the structure of Lenalidomide


Solubility study Applying for BCS based biowaiver is restricted to drug substances performing high solubility and high or low intestinal permeability, i.e., BCS class I and BCS class III, respectively [8]. According to the European Medicines Agency (EMA) guidance, “Lenalidomide is a compound with complete absorption but the available data on solubility does not allow its BCS classification.” [9]. So as to evaluate the API’s aqueous solubility, a study ought to be led to establish the pH-solubility profile of the active substance. In accordance with the ICH guideline, an active substance is categorized as highly soluble if the highest single therapeutic dose is completely solubilized in 250 ml or less of aqueous media at the pH range of 1.2 – 6.8 at 37 ± 1°C [8]. This demonstration requires the investigation in at least three buffers within this range (preferably at pH 1.2, 4.5 and 6.8) and in addition at the pKa if it is within the specified pH range [8]. The equilibrium method was used to demonstrate the API’s solubility, it’s defined by the determination of the dose number, a dimensionless number, calculated as the equation below: . Do=(Dose/S)/(250 ml) Where Do is the dose number, Dose is the highest prescribed dose in mg, S is the solubility of the API (mg/ml) and 250 ml represents the standard volume taken when an oral dosage form is administrated accordingly to the FDA guidance [10]. A dose number equal to or lower than 1signifies high solubility, and a dose number Do > 1 indicates a low- solubility compound [11]. Procedure We debuted the study by investigating in pH: 1.2, 2.3 (pKa), 4.5, and 6.8 with a theoretical concentration of 1.0 mg/ml (100 mg in 100 ml of buffer medium) which represent ten times of the target concentration: 0.1 mg/ml (25 mg in 250 ml, 25 mg is the highest strength of the drug product). The preparation of the buffers was realized in accordance with the European Pharmacopeia recommendations (chapter 5.17.1: Recommendations on dissolution testing). The samples were stirred during five hours using the shake flask method at 37°C. Dissolution study When applying the BCS based biowaiver approach, comparative in vitro dissolution tests ought to be led [8]. In addition to HCL 0.01N dissolution medium, three Pharmacopoeial buffers should be employed: pH 1.2, 4.5, and 6.8. To postulate for a BCS-based biowaiver for BCS class I drug substances, both, reference and test product shall exhibit either rapid or very rapid dissolution rate ≥85% in ≤ 30 min or ≥85% in ≤15min, respectively, also, a comparable in vitro dissolution profiles under all the predetermined conditions. In case that one product has shown a rapid dissolution rate and the other has a very rapid dissolution rate, the statistical similarity must be established. If both products have shown that ≥85% of the label amount of the drug substance is completely dissolved in 15 minutes, statistical evaluation is considered superfluous and the dissolution profiles are viewed as similar [8].


Permeability and absorption Permeability assessment is preferentially based on the extent of absorption obtained from human pharmacokinetic studies, for example, mass balance or absolute bioavailability [8]. High permeability may be appraised when absolute bioavailability is higher or equal to 85% [8]. It can be assessed as well if ≥ 85% of the administered dose is recouped unchanged in urine [8]. In healthy adults and under fasting conditions, the maximum concentration in plasma (Cmax) is observed at 0.77-1.0 h postdose, yet, in patients with AML, the estimated time to attain Cmax is 1.19 h with a standard deviation of 0.55 h after oral administration of  25 mg [4,12, 13]. Higher than 90% of the administered dose is estimated to be absorbed into blood [4]. The primary route of elimination is the urinary excretion where 90% of the dose was recovered unchanged in urine. Insignificant amounts of the drug were excreted in feces (4%) and in semen (0.0062%) [4]. The area under the curve (AUC) is not affected when the drug is co-administrated with food, but it reduces the Cmax [14]. In spite of that, the trials carried out for the pivotal MM registration and for the establishment of the safety and the efficiency of lenalidomide, the drug was administered without taking into consideration the food intake [15]. Distribution Lenalidomide ex-vivo binding to plasma proteins is estimated to be low, for about 40% in healthy adults [16]. After administration of a [14C]-lenalidomide dose, the rate of radioactivity of the latter is estimated to distribute into erythrocytes for about 36-44% which is around the mean hematocrit value estimated to be 42-43%, subsequently, lenalidomide is distributed equally between plasma and cellular component of the whole blood [4], it represents the primary compound in circulation. Hence the plasma represents a suitable vehicle for the distribution of lenalidomide in the whole blood. The range of the apparent volume of distribution of [14C]-lenalidomide is often between 74-91 L in healthy volunteers, which is higher than the total volume of the water in the body (42 L) [12]. Considering the high bioavailability of lenalidomide (> 90 %), it is possible to surmise the existence of a restrained extravascular distribution of the drug substance. As lenalidomide is categorized as a teratogenic drug, the potential of embryo-fetal exposure, transmitted from treated males\’ semen into females of childbearing, must be taken into consideration [6]. Biotransformation and metabolism Lenalidomide is subjected to a fast chiral inversion. The preferred form of lenalidomide in human is the S-form [12]. At 1h postdose of a racemic portion, the R/S concentration ratio in plasma is settled approximately 45 and 55% of the administered dose [12]. After in-vitro studies, it is shown that lenalidomide is neither a substrate of human cytochrome P450 (CYP) enzymes nor a subject to direct conjugative metabolism [17]. Lenalidomide is degraded by a non-enzymatic hydrolysis by dint of the hydrolytic cleavage of the glutarimid ring in hepatocytes at physiological pH, in aqueous solutions and in human plasma showing an in-vitro half-life of roughly 8h [17]. Excretion and elimination For orally administered capsules, lenalidomide is primarily eliminated as unchanged parent drug via renal excretion [6] which is deemed as rapid, due to the short plasma half-life, approximately 3h. Subsequently, it can be inferred that even after repeated doses of lenalidomide, the plasma exhibition does not build up. For healthy adults, 300 ml/min is the total apparent clearance of the drug, 80% of it represents renal clearance and the rest (20%) is accounted for by non-renal clearance [18]. The clearance of lenalidomide is appt to decrease in older healthy volunteers, in spite of that, the renal excretion of the drug is not influenced [16].


Excipients Excipient variations between the reference and the test product must be evaluated for their potential to influence in-vivo absorption. Excipient quantitative and qualitative differences are allowed for BCS class I drug products, in exception of excipients that may influence the drug absorption. In this case, excipients must be quantitatively similar (± 10% of the present amount in the reference product) and qualitatively identical [8]. Excipients used in the reference product are shown in table 2. Table 2: Excipients present in Revlimid 25 mg capsules Excipient Excipient class Anhydrous Lactose Filler Microcrystalline Cellulose Filler Croscarmelose Sodium Disintegrant Magnesium Stearate Lubricant Excipients used in the proposed test product are the same excipients presented in the table above (Table 2). No any excipients that may affect absorption like sugar-alcohols (e.g., mannitol, sorbitol) and surfactants (e.g., sodium lauryl sulfate) were used.


Solubility study The analysis of the samples was done by HPLC, using the QC assay method of the API in the drug product. The results of the solubility study are gathered in the following table (Table 3): Table 3: Results of the solubility study pH 1,2 2,3 (pKa) 4,5 6,8 Maximum Concentration dissolved (mg/ml) 1,01 0,59 0,43 0,34 Final pH of samples 1,26 2,29 3,3 6,79 Do 0.09 0.16 0.23 0.29 Dissolution study Figures 2-5 and table 3 represent the comparative dissolution profiles data of the test product and Revlimid in four dissolution medium, pH 1.2, 4.5 and 6.8 as per the ICH guidelines and HCL 0.01N in accordance with the US FDA dissolution method. Figure 2: Dissolution profile in HCL 0.01N Figure 3: Dissolution profile in pH = 1.2 Figure 4: Dissolution profile in pH = 4.5 Figure 5: Dissolution profile in pH = 6.8 Table 4: Dissolution data of lenalidomide and Revlimid 25 mg capsules Medium Time (min) Lenalidomide % released Revlimid % released HCL 0.01N 0 0 0 10 92 91 15 98 95 20 98 95 30 99 96 45 99 96 pH 1.2 0 0 0 10 98 95 15 99 98 2 100 99 3 100 99 45 100 99 pH 4.5 0 0 0 10 91 98 15 93 99 20 93 100 30 94 100 45 94 100 pH 6.8 0 0 0 10 90 92 15 91 94 20 96 97 30 97 99 45 97 99

DISCUSSION Solubility study The maximum dose strength for lenalidomide is 25 mg. A nominal concentration of 1.0 mg/ml was used to establish the solubility study which represents ten times of the target concentration of 0.1 mg/ml. By using the equilibrium method, the results demonstrated that the compound presents a high solubility in all buffer solutions, confirming that the drug substance is completely dissolved at the target concentration of 0.1 mg/ml. Thus, the dose:solubility ratio is lower than 250 mL and Do ≤1, as indicated in Table 3. The drug classification based on the solubility, shows that 68% of the top 200 US list and 67% of the World Health Organization (WHO) list, are classified as highly soluble drugs based only on the dose number even if the drug has exhibited a solubility inferior to 0.1 mg/ml [11].

Consequently, we can substantiate that the drug substance is highly soluble. Dissolution study In the present study, the dissolution test was performed in four different medium to assess bioequivalence amid the test product and Revlimid. The primary goal of dissolution testing is to use as a qualitative tool to provide the measurement of the bioavailability of a drug. Generic drugs products are duplicates of inventor drug products. They are usually advocated for use because they are typically more affordable than the originator products, ergo enhancing access to life-saving medicaments, particularly in economically developing nations [1]. In case of the present study lenalidomide, 25 mg and Revlimid 25 mg capsules were studied for their bioequivalence studies. First, the dissolution was run in HCL 0.01N (Figure 2). The reference product shows a very rapid dissolution rate, 95% in 15 minutes, whereas, the test product shows a higher dissolution rate, 98% in 15 minutes. Both test and reference products demonstrate that ≥85% of the label amount of the drug is dissolved in 15 minutes, statistical evaluation is unnecessary in this case and the dissolution profiles are considered similar. Also, in pH 1.2, 4.5 and 6.8, (Figures 3-5) all the dissolution rates may be considered as very rapidly dissolving, as more than 85% of the labeled amount of the drug substance dissolve within 15 minutes.

The obtained dissolution patterns are considered similar to the innovator’s patterns in all dissolution test conditions. To sum up, if a drug product undergoes 85% dissolution in 15 minutes under mild dissolution test conditions, generally, then it should not have any bioavailability problems [1]. Pharmacokinetics proprieties Lenalidomide is a compound with complete absorption [9]. The oral absorption of lenalidomide was rapid in the fasted state, with Cmax observed at approximately 1 h. Concomitant administration of lenalidomide with high-fat content meal does not influence the drug exhibition, calculated as AUC, yet it reduces the rate and the extent of the drug absorption, as demonstrated by an estimated 20% decrease in the AUC and an estimated reduction of 50% in Cmax together with a simultaneous delay of 1.6h in Tmax. This delay could be explained by the presence of food in the digestive system, this mild reduction (20%) of lenalidomide oral bioavailability due to food presence is clinically considered irrelevant. The safety and efficacy of lenalidomide were established in pivotal MM and MDS registration trials where the drug was administered without any specific instructions regarding food intake [19, 20, 21].

Thus, it can be inferred that lenalidomide can be taken with or without food. The drug shows a high oral bioavailability (≈90%) that endorses its high permeability. It has a low protein binding, a linear pharmacokinetics, a restricted metabolism and is primarily eliminated by renal excretion as demonstrated in this study [12]. CONCLUSION The main purpose of this study was to evaluate the solubility of lenalidomide in pursuance to prove its BCS class. Based on the obtained results and literature data, lenalidomide is a highly soluble and highly permeable drug substance, so it can be classified as BCS class I. Also, this biowaiver study has shown that our test product complies with the similarity requirements for comparative dissolution testing versus the reference product. We conclude that for lenalidomide 25 mg capsules, granting a biowaiver is justified. This type of bioequivalence studies can reduce the time, cost and unnecessary exposure of healthy subjects to oncology drugs and finally to market quality generic drug products. Acknowledgment Boujbel, et al are employees of Les Laboratoires Medis, which is the sponsor of the biowaiver study for lenalidomide 25 mg capsules. Alsiwisi Mahmoud is an independent pharmacoconsultant working with les Laboratoires Medis. The authors wish to acknowledge the support of Les Laboratoires Medis Research and development Departemen-Oncology team.