Stability behaviour of antiretroviral drugs and their combinations. 9: Identification of incompatible excipients
Moolchand Kurmi, Archana Sahu, Mayurbhai Kathadbhai Ladumor, Arvind Kumar Bansal and Saranjit Singh
A Analytical Research and Development, Biocon Bristol-Myers Squibb Research & Development Center (BBRC), Bangalore 560 099, India.
B Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160 062, Punjab, India.
C Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160 062, Punjab, India.
ABSTRACT
Incompatibility studies of antiretroviral drugs, viz., lamivudine (3TC), emtricitabine (FTC), abacavir sulfate (ABC), tenofovir disoproxil fumarate (TDF), zidovudine (ZDV), efavirenz (EFV) and nevirapine (NVP) were carried out in the presence of ten selected excipients, i.e., microcrystalline cellulose, lactose monohydrate, starch, magnesium stearate, sodium lauryl sulfate, sodium starch glycolate, croscarmellose sodium, colloidal silica, povidone K-30 and hydroxypropyl cellulose. Among all, ABC showed reaction with lactose monohydrate, resulting in the formation of two interaction products, while sodium lauryl sulphate enhanced the degradation of TDF. The interaction products of ABC-Lactose were separated by high performance liquid chromatography (HPLC) and subjected to liquid chromatography-high resolution mass spectrometry (LC-HRMS) studies for their characterization. One of the products was also isolated and subjected to 1D and 2D nuclear magnetic resonance (NMR) studies for structural confirmation. The toxicity of both was predicted using TOPKAT and ADMETTM software and compared to the drug.
INTRODUCTION
Excipients are considered pharmacologically inert, but they can initiate, propagate or participate in chemical and/or physical interactions with the drugs, which may compromise the effectiveness and acceptance of a medication [1]. Therefore, the selection of excipients is vital in the design of a quality drug product. Excipients and their concentration in a formulation are selected, not only based on their functionality, but also on the basis of their compatibility with the drug. Hence drug-excipient compatibility studies are required to be conducted to predict potential of incompatibility in the final dosage form. If the stability of the drug is found to be sacrificed, strategies to mitigate instability of the drug can be adopted. Thus carefully planned and executed drug-excipient compatibility studies can lead to savings in terms of resources and time delays associated with stability issues arising during the late stage of product development [2].
In previous eight parts of this series, we reported intrinsic stability of several antiretroviral drugs, and interaction behaviour among them [3-10]. This ninth part covers the evaluation of drug-excipient compatibility of lamivudine (3TC), emtricitabine (FTC), abacavir (ABC), tenofovir disoproxil fumarate (TDF), zidovudine (ZDV), efavirenz (EFV) and nevirapine (NVP) with the excipients listed by WHO and present in commercial antiretroviral products [11]. In total, ten excipients were taken for the studies, viz., microcrystalline cellulose, lactose monohydrate, starch, magnesium stearate, sodium lauryl sulfate, sodium starch glycolate, croscarmellose sodium, colloidal silica, povidone K-30 and hydroxypropyl cellulose.
2. EXPERIMENTAL
2.1. Chemicals and reagents
All the selected pure drugs were obtained as gratis samples from Aurobindo Pharma Ltd. (Hyderabad, India). Excipients were procured from different sources, viz., microcrystalline cellulose (spray dried, grade PH 101) from DFE Pharma (Bangalore, India); lactose (monohydrate), starch potato and magnesium stearate (precipitate fine powder) from LOBA Chemie (Mumbai, India); sodium lauryl sulfate from s.d. fine-chem Ltd. (Mumbai, India); sodium starch glycolate (explotab) from Penwest Pharmaceuticals Co. (Patterson, NY, USA); croscarmellose sodium from Signet Chemical Corp. Pvt. Ltd. (Mumbai, India); colloidal silica from Evonik Industries (Hanau, Germany); povidone K-30 from ISP Technologies (Wayne, NJ, USA), and hydroxypropyl cellulose (M.W. 1000,000) from Alfa Aesar (Ward Hill, MA, USA). High performance liquid chromatography (HPLC) grade methanol (CH3OH) was purchased from J.T. Baker (Phillipsburg, NJ, USA). Deuterated water (D2O, 99.9%), was purchased from Aldrich (St. Louis, MO, USA). Buffer salts and all other chemicals were of analytical reagent grade. Ultra pure water (H2O) was obtained from ELGA water purification unit (Bucks, England).
2.2. Apparatus and equipments
HPLC studies were carried out on LC-2010C HT liquid chromatograph (Shimadzu, Kyoto, Japan), which was equipped with a SPD-M20A prominence diode array detector. Pursuit XRs LC column (5, C18, 250 4.6 mm, Varian Inc., Lake Forest, CA, USA) was used for analytical studies, while Kingsorb column (5, C18, 250 × 10.0 mm, Phenomenex, Torrance, CA, USA) was used for isolation of the interaction products.
Liquid chromatography-high resolution mass spectrometry (LC-HRMS) studies were carried out using LC-ESI-Q-TOF-MS, in which LC part consisted of 1100 series HPLC (Agilent Technologies, Waldbronn, Germany) that comprised of an on-line degasser (G1379A), binary pump (G1312A), auto injector (G1313A), column oven (G1316A) and a PDA detector (G1315B). The HRMS system consisted of MicrOTOF-Q spectrometer (Bruker Daltonics, Bremen, Germany). System control and data acquisition were done by Hystar software (version 3.1) from the same source. The calibration solution used was 5 mM sodium formate. Nuclear magnetic resonance (NMR) studies were performed on JNM- ECA 500 MHz NMR spectrometer (JEOL, Tokyo, Japan) and the data were processed by Delta v5.0.4.4 software.
pH/Ion analyzer model PB-11 from Sartorius (Goettingen, Germany) was used to check pH of all the solutions. Other equipments used were sonicator (3210, Branson Ultrasonics Corporation, Danbury, CT, USA), precision analytical balance (AG 135, Mettler Toledo, Schwerzenbach, Switzerland) and auto pipettes (Eppendorf, Hamburg, Germany).
2.3. Drug-excipient interaction studies
For these studies, the drug amount in each mixture was fixed as 10 mg. The amount of excipient was decided based on its role in the formulation. The diluents, viz., microcrystalline cellulose, lactose monohydrate and starch (also used as binder), were taken in 1:5 w/w (drug:excipient) ratio. Magnesium stearate (lubricant), sodium lauryl sulfate (lubricant), sodium starch glycolate (disintegrant), croscarmellose sodium (disintegrant), colloidal silica (glidant), povidone K-30 (binder) and hydroxypropyl cellulose (binder) were taken 10 times of their % amount (respective range/2) in the formulation as detailed in Table 1. [12]. The mixtures were weighed into glass vials, followed by addition of 50 l H2O to create maximum humid environment following the protocol proposed by Serajuddin et al. [13]. The same were sealed and stored at 40 C for one month. After completion of the study, all samples were withdrawn and extracted in CH3OH:H2O (50:50 v/v) to a final drug concentration of 2 mg/ml. The samples were analyzed by HPLC. The latter involved mobile phases composed of methanol (A) and 10 mM ammonium formate (pH 3.75) (B) in a gradient mode (Tmin/%B; 0/95; 5/95; 50/10; 55/95; 62/95).
The column oven temperature was 30 C during analysis. The detection wavelength, flow rate and injection volume were 254 nm, 1 ml/min and 5 l, respectively.
2.4. LC-MS studies on ALm1 and ALm2
For LC-HRMS studies on ALm1 and ALm2, the interaction products of ABC and lactose monohydrate, LC method used was similar to the HPLC method employed for analyses of the stability samples. However, to prevent condensation in the ionization source, the solvent flow into MS system was reduced from 1 ml/min to 200 l /min, using a diverter valve. Moreover, in the case of LC-HRMS, instrument parameters were modified suitably to increase the response of interaction, and the same were as follows: end plate offset, -500 V; capillary, 4500 V; nebuliser, 1.2 bar; dry gas, 6.0 L/min; dry temperature, 210 °C; funnel 1 RF, 300 Vpp; funnel 2 RF, 350 Vpp; ISCID energy, 0 eV/z; hexapole RF, 320 Vpp; ion energy, 6 eV/z; low mass, 80 (m/z); collision energy eV/z, 16; transfer time, 120 µs; collision RF, 280 Vpp, and pre pulse storage, 10 µs.
2.5. Additional enrichment, isolation and NMR studies on ALm1
Of the two interaction products, ALm1 was enriched by maintaining the mixture of ABC (200 mg) and lactose monohydrate (400 mg) in the presence of 400 l H2O at 60 °C for 10 d. Its isolation was done on a semi-preparative HPLC column. The mobile phase was methanol (A) and 0.1% H3PO4 (B), which was run in a gradient mode (Tmin/%B; 0/82→7/82→7.01/30→10/30→10.01/82→13/82). The column oven temperature, detection wavelength, flow rate and injection volume were 40 C, 232 nm, 4 ml/min and 90l, respectively. The collected HPLC fraction was dried on a rotary evaporator and the residue was subjected to 1H, 13C, distortionless enhancement by polarization transfer-135 (DEPT-135) and heteronuclear multiple bond correlation (HMBC) NMR studies in D2O. For the purpose of comparison, ABC (1H and 13C) and lactose monohydrate (1H) were also subjected to NMR studies in D2O.
2.6. In-silico toxicity prediction by TOPKAT
Potential toxicity of both the interaction products was predicted by TOPKAT (TOxicity Prediction by Komputer Assisted Technology, Discovery Studio 2.5, Accelrys, Inc., San Diego, CA, USA) [14]. As per the software manual, probability values from 0.0 to 0.30 are advised to be considered as low probabilities for any toxicological end point, values from 0.3 to 0.7 fall in an in-determinate zone, and those greater than 0.70 are considered as high probabilities [15].
2.7. Prediction of ADMET parameters
ADMET Predictor™ software (version 9.0, Simulations Plus, Lancaster, CA, USA) was used for the prediction of physicochemical, and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of the drug and its interaction products. The properties were predicted by physicochemical and biopharmaceutical (PhysChem); metabolism and toxicity modules. The PhysChem module helped to yield ionization (pKa), lipophilicity, solubility, permeability, transporters and pharmacokinetic parameters, and even antiviral inhibition. The metabolism module helps to determine the site of metabolism by nine CYP isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4). Also, the extent of inhibition could be determined for five major drug metabolizing CYP enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4). The toxicity submodels predicted a range of toxicities, including cardiac, hepatotoxicity, endocrine, carcinogenicity, etc.
3. Results and discussion
3.1. HPLC analyses of the stressed drug-excipient mixtures
Figures 1a and b show the comparative chromatograms of ABC and TDF, respectively, in the presence of selected excipients. Evidently, interaction was mainly observed between ABC and lactose monohydrate, resulting in the formation of two novel interaction products, ALm1 and ALm2, apart from previously characterized degradation products A2 (N6-cyclopropyl-9H-purine-2,6-diamine) and A4 (- (2,4-diamino-7H-pyrrolo[3,2-d]pyrimidin-7-yl)cyclopent-2-enyl)methanol) [7]. It was also observed that in the case of TDF, sodium lauryl sulphate accelerated the formation of the drug degradation products T7 (tenofovir monosoproxil methylene adduct), T10 (tenofovir disoproxil ((hydroxymethoxy)methoxy)methyl adduct), T11 (tenofovir mono and disoproxil methylene linked heterodimer) and T12 (methylene linked dimer of tenofovir disoproxil) identified in a previous study from our laboratory [3], owing perhaps to the wetting property of sodium lauryl sulphate. The behaviour of ZDV, FTC, EFV and NVP was not altered in the presence of excipients.
3.2. Characterization of the interaction products ALm1 and ALm2
The two Interaction products, ALm1 and ALm2, were characterized by LC-HRMS data and the structure of ALm1 was additionally verified through 1D and 2D NMR studies. LC-HRMS spectra of both the products are shown in Fig. 2. Their accurate and exact masses, best molecular formulae, errors in mmu, and m/z of their fragments are listed in Table 2.
3.2.1. Interaction product ALm1
The molecular ion of ALm1 had accurate and exact masses of 611.2607 and 611.2671 Da, respectively (error = -6.4 mmu), for which, the molecular formula worked out to be C26H39N6O11+. It was identified as Maillard product formed on interaction of primary amine of ABC and hydroxyl group of lactose. Its structure and fragmentation behaviour, which are shown in Fig. 3, highlighted that molecular ion of ALm1 fragmented into product ions of m/z 593, 515 and 449 on loss of H2O, C6H8O and C6H10O5, respectively. The precursor ion of m/z 593 dissociated into fragments of m/z 575 and 329 on sequential losses of H2O and C10H14O7, respectively. Also, the precursors of m/z 515 and 449 reduced into ions of m/z 497 and 431, on loss of H2O in both the cases. The latter ion formed fragment of m/z 413 on loss of H2O, which further reduced into ion of m/z 287 on loss of C6H6O3. The molecular ion of ALm2 also dissociated into fragment of m/z 353 and 191 (also formed from precursor of m/z 287) on sequential losses of C6H8O and C6H10O5, respectively.
For further confirmation of ALm1 structure, NMR data were obtained for ABC, lactose and ALm1 in D2O. Their numbered structures are shown in Fig. 4. Comparative proton spectra of ABC, lactose and ALm1 are shown in Fig. 5 and 13C spectra of ABC and ALm1 are shown in Fig. 6, while DEPT-135 and HMBC spectra of ALm1 are given in Figs. 6b and 7, respectively.
As shown in Fig. 5, signals for all the protons of ABC (Fig. 5a) and lactose (Fig. 5b) appeared in the case of ALm1 (Fig. 5c) with very minor change in chemical shift values. 13C and DEPT-135 (Fig. 6a and b) data also did not show any significant change in chemical shift values of carbon signals corresponding to 13C data of ABC (Fig. 6c). The structure was substantiated by HMBC data (Fig. 7), which clearly showed correlation between H-1’ and C-2, which confirmed the connectivity between ABC and lactose.
3.2.2. Interaction product ALm2
Accurate mass of this product was determined to be 449.2153 Da with 1.0 mmu error from its exact mass i.e., 449.2143 Da (which was also a fragment of ALm1). The molecular formula for the same was determined to be C20H29N6O6+. Its structure and fragmentation pattern is shown in Fig. 3.The molecular ion fragmented into ion of m/z 431 and 353 on loss of H2O and C6H8O, respectively. The precursor ion of m/z 431 on sequential losses of H2O, C6H6O3 and C6H8O dissociated into fragments of m/z 413, 287 and 191, respectively. The latter ion was also formed from precursor of m/z 353 on loss of C6H10O5. The parallelism of fragments of ALm2 and ALm1 indicated that it was related to the latter.
3.3. Interaction between ABC and lactose leading to formation of ALm1 and ALm2
The interaction pathway leading to ALm1 and ALm2 is shown in Fig. 8. Evidently, ALm1 was formed by Maillard reaction between free amino group of ABC and hydroxyl group of lactose on loss of H2O. It also shows formation of ALm2 upon glycosidic bond hydrolysis of ALm1, with release of single hexose molecule in the process.
3.4. In-silico toxicity of interaction products ALm1 and ALm2 by TOPKAT
The results of TOPKAT analysis of the drug, ALm1 and ALm2 are given in Table 3. The two interaction products showed nearly similar toxicity profile. Difference was mainly in the cases of FDA carcinogenicity female rat and developmental toxicity potential, where ALm1 showed high risk of toxicity in comparison to ABC as well as ALm2. Oppositely, potential for Ames mutagenicity was higher in the case of ALm2. Rat maximum tolerated dose was also near to zero for both, which highlighted their high toxicity potential. FDA carcinogenicity female mouse single vs multiple (v3.1) and weight of evidence carcinogenicity call (v5.1) toxicities were also predicted to be higher for both the interaction products with respect to the drug.
3.5. ADMET predictor™ results
The results of in silico ADMET predictions are summarized in Table 4. The predicted aqueous solubility of both the interaction products was same, but more than the parent drug. Predicted octanol-water partition coefficient (log P) values, human effective jejunal permeability (Peff), and volume of distribution (Vd) in humans at steady state for ALm1 and ALm2 were lower in comparison to the drug.
The drug as well as its interaction products was predicted to have inhibitory potency against CYP3A4. However, the interaction products were revealed to be nonsensitizers, against sensitizing potential of the drug. All three were predicted to be free of cardiac and reproductive toxicity.
4. Conclusion
The study revealed drug-excipient interaction among ABC and lactose monohydrate was because of Maillard reaction between amino and hydroxyl group of ABC and lactose, respectively, leading to two products (ALm1 and ALm2). It also highlighted that sodium lauryl sulfate hasten the formation of degradation products (T7 and T10- T12), which were formed on interaction with formaldehyde. This could be because of enhanced contact of drug molecules and better penetration of formaldehyde in reaction mixture in presence of SLS. The two interaction products were characterized using LC-MS, and in case of ALm1, additional NMR data was also generated for support. Toxicity potential of formed products was assessed using TOPKAT, while ADMET properties were also predicted. The investigation highlighted the absence of incompatibility of 3TC, FTC, ZDV, EFV and NSC 641530 with the ten selected excipients, i.e., microcrystalline cellulose, lactose monohydrate, starch, magnesium stearate, sodium lauryl sulfate, sodium starch glycolate, croscarmellose sodium, colloidal silica, povidone K-30 and hydroxypropyl cellulose.