Forced Degradation of an Anticancer Drug Apalutamide: Impurity Profiling and Structure Elucidation Study

doi:10.21203/rs.3.rs-3309259/v1

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Research Article

Forced Degradation of an Anticancer Drug Apalutamide: Impurity Profiling and Structure Elucidation Study

https://doi.org/10.21203/rs.3.rs-3309259/v1

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published 09 Nov, 2023

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An anticancer drug apalutamide approved by USFDA in 2018 is one of the most commonly prescribed chemotherapeutic agents used for the treatment of prostate cancer. The present study aims at establishing the chemical structures of all the degradation products of the API formed under different stress conditions. Apalutamide degradation was studied under ICH-recommended conditions of hydrolysis (acidic, alkaline, neutral), oxidation, photolysis, and thermal stress. In total, seven degradation products (DP-1 to DP-7) were observed which were successfully separated on HPLC and further characterized using LC-MS/MS Q-TOF. The chromatographic separation was achieved using a Shimpack C18 column (4.6 mm × 250 mm, 5 µm) with 0.1% formic acid and acetonitrile as a mobile phase in a gradient elution mode. The injection volume, detection wavelength, and flow rate were set at 10 µL, 240 nm, and 1.0 mL/min, respectively. Degradation pathways for each of the observed DPs have been proposed based on the mass fragmentation pattern of API as well as DPs, identifying the underlying chemical transformations. Prediction of DPs was performed with Zeneth software tool and the results were compared with the experimental observations. In silico toxicity assessment carried out using Derek suite reveals toxic nature of some of these DPs.

Apalutamide

forced degradation

stability

LC-MS/MS

Apalutamide (APA; Fig. 1) is a non-steroidal antiandrogen developed for the treatment of prostate cancer by Janssen Biotech, Inc. and received its first global approval in the USA for the treatment of non-metastatic castration-resistant prostate cancer (nmCRPC) in February 2018 [1–3]. APA acts by blocking the ligand binding domain of the androgen receptor. Chemically, it is e (4-(7-(6-cyano-5-(trifluoromethyl) pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro [3.4] octan5-yl)-2-fluoro-N-methylbenzamide) with molecular formula C₂₁H₁₅F₄N₅O₂S. It is sold under the brand name Erleada™ [3, 4].

Regulatory requirements and product quality are the top priorities of pharmaceuticals. During storage, the appearance of impurities in active ingredient and formulations can lead to product recalls which can cause a huge loss to the manufacturing companies. In order to deal with this situation prior knowledge of drug stability is important, which can be obtained by performing stress degradation studies in accordance with ICH Q1 R2 and Q1B guidelines. The stability of the drug is analyzed against high temperature, high and low pH extremes, oxidation, and photolytic conditions. These studies provide the data of stability of drug as well as an illustration of degradation pathways [5, 6].

Several analytical and bioanalytical methods have been reported for the determination of APA, its related impurities, and metabolites [7–15]. Bandaru et. al. conducted a study focused on the development and validation of an HPLC method for the analysis of apalutamide-related substances in solid dosage forms [9]. In addition, De Vries et. al. reported a metabolite study of APA using LC-MS [15]. In one of the reports, a stress degradation of the API was attempted however, it was limited to development of stability indicating HPLC based method, and did not provide any information on the chemical structures of the degradation products [8].

The present work aims at (i) Study of stress degradation behavior of APA; (ii) Development of HPLC method for separation of its degradation products; (iii) Characterization of degradation products by LC-MS/MS Q-TOF and NMR (iv) Mechanistic understanding on degradation pathways, and (v) Toxicological risk assessment, using in silico prediction.

2.1. Chemicals and Reagents

APA was obtained from Aurobindo Pharma Ltd., India as a gift sample. LC–MS grade acetonitrile (ACN) was procured from Sigma-Aldrich, Bangalore, India (BIOSOLVE®). HPLC grade ACN was purchased from Duksan Pure Chemicals Co. Ltd., South Korea. Formic acid (HCOOH) and sodium hydroxide (NaOH) were procured from Sisco Research Laboratories Pvt. Ltd., India. AR-grade H₂O₂ was obtained from Finar Ltd., India. Ultra-pure grade water was acquired from Ultra Clear TWF system by Evoqua Water Technologies LLC, USA.

2.2. Instrumentation and equipments

Alliance high performance liquid chromatography system-e2695 (Waters Corp., Milford, MA, USA) along with a photodiode array (PDA) detector (model 2998) was used for the analysis of stress degradation samples. Mass studies were performed by using Agilent 1200 series LC instrument attached to a quadrupole time-of-flight mass spectrometer (Q-TOF LC/MS 6540 series, Agilent Technologies, USA). For structural characterization elemental composition calculator (Frank antolasic) was used throughout mass analysis. Preparative HPLC (Waters, MA, USA) equipped with a UV/Visible detector (Waters 2489) was used for isolation of DP-7. NMR experiments performed on Bruker 500 MHz (AVANCE III HD-500, Switzerland)) using solvent DMSO-d6 and internal references standard Tetramethylsilane. A photostability chamber (Newtronic, lifecare equipment Pvt. Ltd., India) with fluorescent and ultraviolet (UV) lamps was used to execute the photostability studies as specified in the ICH guideline Q1B. An ultra-sonicator (PCI Analytes) and weighing balance (Sartorius, QUINTIX224-10IN, Maharashtra) were used for sample preparations. Hot plate (IKA RCT Basic, Bengaluru, India) with oil bath was employed for executing the solution phase degradation study at 80°C.

2.3. Forced degradation study

A forced degradation study was performed according to ICH Q1(R2) [16]. Water and acetonitrile (1:1% v/v) was used as diluent because of poor solubility of APA in water. A forced degradation study was carried on a 500 ppm solution of APA. Acid and neutral degradation studies were carried out in 2N HCl, and diluent for 24 hr and 5 days respectively at 80°C. Alkaline degradation was performed on 0.1N NaOH at room temperature. An oxidation study was executed in 3% H₂O₂ peroxide for 3 hours in the dark condition. Seven-day dry heat exposure of solid powder of APA in a Petri plate at 80°C led to a thermal degradation experiment in the solid state. The photolytic stress study was conducted at 25°C by exposing a solution and solid sample to 200 Whm^− 2 of UV illuminations and 1.2 × 10 million lux hr of fluorescent light.

2.4. Sample preparation

The 500 µL of stressed samples were collected at specified time points. A sufficient concentration of NaOH and HCl was used to neutralize the hydrolysis samples of acid and base respectively. Samples from neutral, oxidative, and photolytic stress condition were diluted with the diluent. In the solid-state study, a sample was dissolved using the diluent. A 250 ppm was the final sample concentration injected in HPLC. All the above solutions were filtered using syringe filter 0.22 µm.

3.2. Isolation and enrichment of DP-7

DP-7 was formed as a major degradation product in acid hydrolysis. Isolation of DP-7 was carried out using a preparative HPLC column, Xbridge (19 mm × 300 mm, 5 µm). The mobile phase was used as 10 mM ammonium acetate (solvent A) and acetonitrile (solvent B) for optimum separation of DP-7 and the drug. The flow rate was set as 10 mL/min. The gradient condition was T_min/%B: 0/10, 3/10, 18/80, 23/80, 30/10. A 240 nm wavelength was used for detection. By using a rotary evaporator, the collected fraction was evaporated.

2.5. Characterization of forced degradation products

LC-MS/MS Q-TOF technique with mass hunter acquisition software from Agilent was used for the characterization of degradation products. All DPs except DP-7 were run in positive ionization mode. For DP-7, negative ionization mode was used. A calibration of mass spectrometry was performed by using a standard tuning mixture. The parameter for mass spectrometry for drying gas, capillary voltage, fragmentor voltage, skimmer voltage, collison energy etc. was optimized to get the best results. All optimized parameters are mentioned in Table 1. All data in mass spectrometry was acquired in the range m/z 50-1000 in auto MS/MS mode. Plausible chemical structures of the DPs and their proposed fragments were proved using elemental composition calculator, RDB (ring and double bond equivalent) value with minimum mass error between observed and theoretical mass (Table S1). The recorded NMR spectra of APA and DP-7 were analysed and plotted in Topspin 4.1.4.

Table 1

LC-MS/MS parameters
Parameters	Positive ion mode	Negative ion mode
Gas temperature	325°C	325°C
Drying gas	12 L/min	12 L/min
Nebulizer	40 psig	40 psig
Voltage capillary	3500 V	3500 V
Fragmentor	140 V	120 V
Skimmer	65 V	65 V

2.6. In silico toxicity prediction tools

In silico degradation products prediction was carried out using Zeneth software (version 8). Derek software (Program version Derek nexus: 6.2.0, Nexus:2.5.0) was used to carry out in silico toxicity of APA degradation products. Human was selected as a specimen for the study.

3.1. Method development and optimization

During the method development, different columns such as Xbridge C18 (4.6 x 250 mm, 5 µm), Xbridge C18 (4.6 x 150 mm, 3.5 µm), Alpha adhoc C8 (4.6 x 250 mm, 5 µm), Shimpack solar C18 (4.6 x 250 mm, 5 µm) were used with a mobile phase of 0.1% formic acid (mobile phase A) and ACN (mobile phase B). A good resolution was observed in Shimpack solar C18 (4.6 x 250 mm, 5 µm) column. Optimal separation of degradation products was achieved by using gradient mode of T_min/%B: 0/10, 3/10, 18/80, 23/80, 30/10. The flow rate and injection volume was set as 1 mL/min and 10 µL respectively. A detection wavelength 240 nm was used throughout the analysis. The column compartment and sampler temperature were 30°C and 25°C respectively. The same method was transferred to LC-MS using T union with a total flow rate of 0.5 mL/min.

3.3. Degradation of APA

A HPLC-PDA was used to monitor the degradation behavior of APA under a variety of stress degradation conditions. Optimum forced degradation was observed under hydrolysis (acid, base, neutral) and oxidative conditions whereas, the compound was found to be stable in photolysis and thermal condition. A total of seven DPs (DP-1 to DP-7) were observed and characterized by LC-ESI-MS/MS. The optimized forced degradation conditions of APA are given in Table 2. Figure 2 represents an overlay of HPLC chromatograms obtained from the forced degradation study.

Table 2

Optimized forced degradation conditions of apalutamide
Stress condition	Concentration of stressor	Exposed condition	Duration	Degradation products formed
Acid hydrolysis	2N HCl	80°C	24 hr	DP-4, DP-6, DP-7
Base hydrolysis	0.1N NaOH	RT	12 hr	DP-4, DP-7
Neutral hydrolysis	H₂O	80°C	5 days	DP-2, DP-3, DP-4
Oxidation	3% H₂O₂	RT	3 hr	DP-1, DP-3, DP4, DP-5, DP-6
Photolysis (Solid state and solution state)	NA	UV and fluorescent light	15 days	Not formed
Thermal (Solid state)	NA	80°C	7 days	Not formed

3.4. Structural characterization of degradation products

Structures of all the degradation products corresponding to each of the peaks observed in the chromatograms were elucidated based on the HRMS data obtained from LC-MS/MS experiments. The mass fragmentation pattern of APA was first studied and molecular formula for each of the fragment was assigned with high accuracy. The MS/MS patterns obtained for each of the DPs were assigned and their molecular structures were elucidated. Mass fragmentation pattern of APA is described below along with highlights from the MS/MS patterns of all the DPs leading to their structure determination. The LC-MS/MS spectra and mass fragmentation data of the DPs are provided in the supplementary information (Fig. S2 - Fig. S15).

3.4.1 Mass spectrometric fragmentation of apalutamide

For better understanding of the fragmentation pathway of APA ([M + H]⁺ 478.0957), its MS/MS spectra was analyzed. All obtained m/z values of fragments were analyzed for plausible chemical structure using an elemental composition calculator and compared with theoretical masses for minimum mass error in ppm. The product ion with m/z 450 was formed due to elimination of ethylene moiety from the unsaturated cyclobutane ring. The product ion at m/z 419 was formed due to loss of methyl amine from m/z 450 which further fragmented into m/z 391 with the elimination of carbon monoxide (CO). The product ion peak at m/z 206 was formed due to cleavage of diazaspiro ring leaving (4-(N-ethynylmethanethioamido)-2-fluorobenzylidyne)oxonium cation which again fragmented into m/z 162 by loss of CS. The product ion peak at m/z 249 was formed with the elimination of C₈H₂F₃N₃S from protonated drug, which further fragmented into m/z 221 due to loss of carbon monoxide (CO). The product ion peak at m/z 58 denotes the presence of N-methyl formamide in drug structure. Other product ion peaks were observed at m/z 432 (loss of H₂O from m/z 450), m/z 175 (loss of C₉H₂F₃N₃OS from m/z 432), m/z 136 (loss of C₂HN from m/z 175), m/z 193 (loss of C₉H₂F₃N₃OS from m/z 450) and m/z 156 (loss of C₁₁F₃N₃O₂ from m/z 419). Figures 3 and 4 show the mass spectrum and proposed fragmentation pathway of APA, respectively. The LC-MS/MS data of APA for plausible structures, RDB value and minimum error in ppm are summarized in Table 3.

Table 3

LC-MS/MS data of apalutamide
Best possible molecular formula	Experimental mass	Exact mass of most probable structure	RDB	Error in ppm
C₂₁H₁₆F₄N₅O₂S⁺	478.0957	478.0955	14.5	0.42
C₁₉H₁₂F₄N₅O₂S⁺	450.0646	450.0642	14.5	0.89
C₁₉H₁₀F₄N₅OS⁺	432.0540	432.0537	15.5	0.69
C₁₈H₇F₄N₄O₂S⁺	419.0224	419.0220	15.5	0.95
C₁₇H₇F₄N₄OS⁺	391.0272	391.0271	14.5	0.26
C₁₃H₁₄FN₂O₂⁺	249.1033	249.1034	7.5	-0.40
C₁₂H₁₄FN₂O⁺	221.1086	221.1085	6.5	0.45
C₁₀H₅FNOS⁺	206.0060	206.0070	8.5	-4.85
C₁₀H₁₀FN₂O⁺	193.0773	193.0772	6.5	0.52
C₁₀H₈FN₂⁺	175.0669	175.0666	7.5	1.71
C₉H₅FNO⁺	162.0351	162.0350	7.5	0.62
C₇H₇FNS⁺	156.0277	156.02782	9.5	-0.64
C₈H₇FN⁺	136.0558	136.0557	5.5	0.73
C₂H₄NO⁺	58.0291	58.0287	1.5	6.89

3.4.2 DP-1

Oxidative condition led to the formation of DP-1. The molecular ion of DP-1 was displayed in positive ionization mode at m/z 295.1082. Carbon monoxide is lost from the aldehyde group of the parent ion to form the product ion at m/z 267. It was found that the product ions identified at m/z 249, m/z 221 and m/z 193 were similar to those of the drug APA while the molecular fragment corresponding to m/z 236 is formed owing to loss of methyl amine. Removal of C₂H₄ from m/z 267 led to the formation of product ion at m/z 239. From m/z 239 loss of methyl amine resulted in a molecular ion at m/z 208. From the observed fragmentation pattern the proposed structure for DP-1 is 1-(N-(3-fluoro-4-(methylcarbamoyl)phenyl)formamido)cyclobutane-1-carboxylic acid.

3.4.3. DP-2

The DP-2 was formed in neutral hydrolysis condition. The protonated mass of DP-2 was m/z 267.1141 and has mass difference 211 Da from protonated drug (m/z 478). The calculated elemental composition obtained by exact mass measurement corresponds to the addition of one O atom and loss of C₈F₃N₃S from APA. The product ions m/z 249, m/z 221, m/z 193, m/z 162 m/z 136, m/z 58 were found common to drug product ions. DP-1 and DP-2 shared the product ions at m/z 239, m/z 236, m/z 208. By elimination of carbon monoxide from product ion at m/z 162, a fragment at m/z 134 was formed. Based this mass fragmentation pattern, the structure proposed for DP-2 is 1-((3-fluoro-4-(methylcarbamoyl)phenyl)amino)cyclobutane-1-carboxylic acid.

3.4.4. DP-3

DP-3 was observed in neutral hydrolysis condition. The mass spectra of DP-3 displayed the [M + H]⁺ ion at m/z 188.0425. The fragment having m/z 171 was observed due to the loss of ammonia. The loss of hydrogen fluoride resulted in the formation of a product ion at m/z 168. The product ion formed at m/z 161 due to removal of HCN which further dissociated into m/z 141 with the elimination of HF. The appearance of fragment ion at m/z 68 indicates the detachment of trifluoromethyl cation. Based on the observed fragments, the structure of DP-3 is proposed to be A 5-amino-3-(trifluoromethyl)picolinonitrile.

3.4.5. DP-4

DP-4 was formed in hydrolysis (acid, alkaline, and neutral) and oxidative conditions with accurate experimental mass of m/z 496.1057. The mass difference between parent protonated drug (m/z 478) and DP-4 is 18 Da which indicates hydration of cyanide group. Major product ions were observed at m/z 479, m/z 423, m/z 392 and minor product ions detected at m/z 468, m/z 451, m/z 423, m/z 58. The product ion of m/z 479 which was formed from DP-4 due to loss of NH₃ confirmed the presence of a terminal amide group in the structure. The fragment ion at m/z 468 was formed by elimination of ethylene moiety from the fused cyclobutane ring in a similar way as it was seen for the drug APA. This ion was further fragmented into m/z 451 by loss of ammonia. The daughter ion formed from m/z 451 showed m/z at 423 indicating the loss of CO. The m/z 58 ion was also found as a common minor fragment with the drug APA. The fragment at m/z 423 results from the loss of C₂H₃NO₂ from the parent ion which further generated a fragment at m/z 392 with the elimination of CH₅N. From above information, the structure of DP-4 is assigned as 5-(5-(3-fluoro-4-(methylcarbamoyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinamide.

3.4.6. DP-5

DP-5 with protonated molecular ion at m/z 464.1326 was formed in oxidative condition. DP-5 dissociated into fragment at m/z 433 with loss of CH₅N. The ion at m/z 433 further fragmented to m/z 405 due to the removal of carbon monoxide. The product ion at m/z 377 was observed with the elimination of C₂H₄. Due to breaking of the central amide linkage of the protonated DP-5, the product ion at m/z 277 was observed. The product ions at m/z 249, m/z 221, m/z 162, m/z 58 were found to be common with drug’s fragment ions. The fragment at m/z 138 was due to loss of C₁₂H₈F₃N₃ from ion at m/z 405. Based on the fragments observed, mechanistic understanding of the degradation pathway (described in section 4) and associated evidence from literature[17], the proposed structure of DP-5 is 4-(N-(1-((6-cyano-5-(trifluoromethyl)pyridin-3-yl)carbamoyl)cyclobutyl)formamido)-2-fluoro-N-methylbenzamide.

3.4.7. DP-6

DP-6 was formed in oxidative condition as a major degradation product. The protonated form of DP-6 showed [M + H]⁺ ion at m/z 462.1176. DP-6 exhibits a mass difference of 16 Da with respect to the parent protonated drug, indicating a replacement of sulfur by oxygen. The product ions observed for this DP at m/z 434, m/z 416, m/z 403 were formed in similar way to those noted in case of APA, with the sulfur replaced by the oxygen. The m/z 431 was observed with the loss of methyl amine from protonated DP. The fragment ion at m/z 377 formed due to removal of C₂H₃NO. The product ions at m/z 249, m/z 221, m/z 193, m/z 162, m/z 136 and m/z 58 exhibited commonality with the drug fragments, as these fragments are related to the terminal groups of the API. From above data, the structure of DP-6 is proposed to be an oxidative desulfuration product of APA, chemically known as 4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-6,8-dioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide.

3.4.8. DP-7

DP-7 was formed in acidic and alkaline hydrolysis conditions. In contrast to the other DPs, ionization of DP-7 could not be achieved in positive mode despite several attempts. The molecular ion peak was found at m/z 463.0484 for [M-H]⁻ ion in the negative ionization mode. The product ion formed at m/z 419 due to loss of CO₂ which further fragmented into m/z at 391 with the elimination of C₂H₄. The fragment ions with m/z 371 (loss of HF) and m/z 297 (loss of C₆H₂) were observed. Based on the mass data the proposed structure of DP-7 is 4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluorobenzoic acid, which is in alignment with the NMR data acquired on the isolated fraction (see supplementary information).

Chemical structures of the proposed degradation products are shown in Fig. 5 below. Interestingly, four (DP-2, DP-4, DP-6 and DP-7) of these seven degradation products observed in this study are also metabolites of APA[15].

All the seven observed degradation products of the API seem to result from feasible chemical transformations depending on the experimental conditions employed. DP-4 and DP-7 are simple hydrolyzed products due to hydrolysis of the nitrile group and the terminal amide group, respectively [18]. DP-6 results from an oxidative desulfuration reaction, formed as a major product in peroxide mediated oxidation condition, and as a minor product under acid hydrolysis condition at high temperature (80°C) [19]. Formation of DP-5 is affected by an attack of peroxide moiety and loss of sulfur dioxide molecule followed by hydrolysis and a ring opening reaction [20, 21]. DP-1 is an amide hydrolysis product of DP-5. DP-2 results from a loss of thioformaldehyde from the diazospirooctanyl ring followed by hydrolysis. DP-3 is formed as a side product from the hydrolysis of the central amide group of the API resulting in the formation of DP-2. It is also a secondary degradation product resulted from the hydrolysis reaction of DP-5. Detailed reaction pathways for the formation of all the DPs are provided in Supplementary Information (Fig. S19).

5.1 Predicted vs. Observed DPs

Prediction of possible degradation products of APA was performed by providing the input conditions corresponding to each of the stress conditions employed experimentally. While closely resembling input conditions were fed with respect to the actual reaction condition, the medium of reaction was chosen as water due to limitations with the available input options in the software. Overall, 13 degradation products were predicted (5 primary DPs and 8 due to further degradation of primary DPs). Structures of the predicted DPs along with other relevant details are provided in the supplementary information (Table S2). Surprisingly, only 2 of the 7 experimentally observed DPs were predicted by the in-silico approach − these are obvious transformation products resulting due to hydrolysis of nitrile and amide groups of the API. It is to be noted that the prediction algorithm was unable to predict DPs resulting from complex reactions like oxidative desulfuration and ring opening of diazospirooctanyl moiety. The contrasting observations between the experimental study and the in-silico assessment underscore the importance of experimental studies for real time assessment of stability of the products.

5.2 In silico toxicity study

Table 4 represents the plausible toxicity of all degradation products for carcinogenicity, pulmonary toxicity, and androgen receptor modulation. A DP-3 (m/z 188) showed carcinogenicity effect in humans’ model due to alert of aromatic amine or amide. DP-4 (m/z 496) and DP-7 (m/z 463) showed pulmonary toxicity due to presence of aromatic thiourea. DP-6 (m/z 462) and DP-7 (m/z 463) are acting as androgen receptor modulation same as APA. All remaining DPs showed inactive actions toward carcinogenicity, pulmonary toxicity, and androgen receptor modulation.

Table 4

*In silico* toxicity predication of DPs by Derek prediction
Sr no	DPs	Carcinogenicity in human	Androgen receptor modulation	Pulmonary toxicity in human
1	DP-1	Inactive	Inactive	Inactive
2	DP-2	Inactive	Inactive	Inactive
3	DP-3	Plausible (Alert Aromatic amine or amide)	Inactive	Inactive
4	DP-4	Inactive	Inactive	Plausible (Alert Aromatic thiourea)
5	DP-5	Inactive	Inactive	Inactive
6	DP-6	Inactive	Plausible (Alert 4-(2,5-Dioxopyrrolidin-1-yl)benzonitrile or analogue)	Inactive
7	DP-7	Inactive	Plausible (Alert 4-(2,5-Dioxopyrrolidin-1-yl)benzonitrile or analogue)	Plausible (Alert Aromatic thiourea)

Forced degradation study of an anticancer drug apaluatamide reveals the occurrence of seven API related impurities (DP-1 to DP-7) that arise due to the degradation of the compound under common stress conditions such as oxidation, hydrolysis (acid, base, and neutral). Each of the seven impurities were separated on the chromatographic system with excellent peak profiles. The chemical structures of all seven impurities were determined based on their HRMS data and thorough MS/MS pattern assignments. Degradation pathways were established for each of the observed DPs indicating risks associated with the exposure of API to respective stress conditions. Interestingly, four of the seven degradation products (DP-2, DP-4, DP-6, and DP-7) were also found to be metabolites of the API in human[15]. Prediction of DPs using Zeneth software suggested the formation of several DPs however, only 2 of the 7 experimental DPs were found to be common. In silico toxicity assessment carried out using the Derek suite suggests the carcinogenic nature of DP-3, pulmonary toxicity of DP-4 and DP-7 along with androgen receptor modulation potential of DP-6 and DP-7. This indicates the need to further study experimental toxicity of DP-3 as well as biological activity of metabolites DP-6 and DP-7 in comparison with APA. Knowledge about the chemical stability of the API with respect to exposure to different environments will be useful to adopt mitigation strategies to avoid and control degradation related impurities in the drug product.

Apalutamide API ACN	APA Active pharmaceutical ingredient Acetonitrile
DP	Degradation product
HPLC	High performance liquid chromatography
HCl	Hydrochloric acid
ICH	International Council for Harmonisation
LC-MS/MS	Liquid chromatography-mass spectrometry/ mass spectrometry
NaOH	Sodium hydroxide
ND	Not detected
NMR	Nuclear magnetic resonance
RT	Room temperature

Acknowledgements

The authors are thankful to National Institute of Pharmaceutical Education and Research, Hyderabad, India for providing facilities to perform this research work (NIPER communication number: NIPERHYD/2023/69). AGD thanks SERB-DST, Government of India for the Start-up Research Grant (SRG/2022/001553).

Compliance with Ethical Standards

Conflicts of Interest

The authors have no conflict of interest.

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No competing interests reported.

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published 09 Nov, 2023

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Forced Degradation of an Anticancer Drug Apalutamide: Impurity Profiling and Structure Elucidation Study

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Abstract

Figures

1. Introduction

2. Experimental

2.1. Chemicals and Reagents

2.2. Instrumentation and equipments

2.3. Forced degradation study

2.4. Sample preparation

3.2. Isolation and enrichment of DP-7

2.5. Characterization of forced degradation products

2.6. In silico toxicity prediction tools

3. Results and discussions

3.1. Method development and optimization

3.3. Degradation of APA

3.4. Structural characterization of degradation products

3.4.1 Mass spectrometric fragmentation of apalutamide

3.4.2 DP-1

3.4.3. DP-2

3.4.4. DP-3

3.4.5. DP-4

3.4.6. DP-5

3.4.7. DP-6

3.4.8. DP-7

4. Mechanism of formation of DPs

5. studies for prediction and toxicity assessment of DPs

5.1 Predicted vs. Observed DPs

5.2 In silico toxicity study

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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