Olaparib Research Articles

Novel drug-drug co-amorphous systems of olaparib with nonsteroidal anti-inflammatory drugs with improved solubility, physical stability, antitumor activity and pharmacokinetics

Olaparib (OLA), the first FDA-approved PARP inhibitor, has been approved successively for monotherapy or maintenance treatment of ovarian, breast and pancreatic cancer. Unfortunately, the poor solubility and bioavailability of OLA have limited its application. In this study, novel drug-drug co-amorphous systems of OLA with five nonsteroidal anti-inflammatory drugs (NSAIDs), including flurbiprofen (FLU), ketoprofen (KET), ibuprofen (IBU), diclofenac (DIC) or indomethacin (IND), were prepared for improving the aqueous solubility of OLA and achieving combination therapy against ovarian cancer. These new co-amorphous products were confirmed by powder X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared spectroscopy. Five co-amorphous systems were more soluble and released faster than the crystalline OLA in pH 6.8 buffer, and OLA-DIC and OLA-IND showed superior physicochemical stability under accelerated storage conditions. The antitumor activities of OLA-FLU, OLA-KET, OLA-IBU and OLA-DIC against OVCAR3 cell line were approximately increased by 3.5, 2.4, 2.7, and 6.0-fold than that of OLA, respectively. Moreover, pharmacokinetic studies of co-amorphous OLA-DIC in rats revealed that the OLA exhibited 6.14-fold improvement in AUC0-t value compared with crystalline OLA. Therefore, our study suggested that OLA-NSAIDs co-amorphous systems showed the great potentials to improve the solubility, dissolution behaviors, and bio-pharmaceutical performance of OLA.

Investigation of Stabilized Amorphous Solid Dispersions to Improve Oral Olaparib Absorption.

In this study, we investigated the formulation of stable solid dispersions to enhance the bioavailability of olaparib (OLA), a therapeutic agent for ovarian cancer and breast cancer characterized as a BCS class IV drug with low solubility and low permeability. Various polymers were screened based on solubility tests, and OLA-loaded solid dispersions were prepared using spray drying. The physicochemical properties of these dispersions were investigated via scanning electron microscopy (SEM), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Fourier Transform Infrared Spectroscopy (FT-IR). Subsequent dissolution tests, along with assessments of morphological and crystallinity changes in aqueous solutions, led to the selection of a hypromellose (HPMC)-based OLA solid dispersion as the optimal formulation. HPMC was effective at maintaining the supersaturation of OLA in aqueous solutions and exhibited a stable amorphous state without recrystallization. In an in vivo study, this HPMC-based OLA solid dispersion significantly enhanced bioavailability, increasing AUC0-24 by 4.19-fold and Cmax by more than 10.68-fold compared to OLA drug powder (crystalline OLA). Our results highlight the effectiveness of HPMC-based solid dispersions in enhancing the oral bioavailability of OLA and suggest that they could be an effective tool for the development of oral drug formulations.

Electrochemical and Theoretical Investigations on the Binding of Anticancer Drug Olaparib to Human Serum Albumin

Abstract This article explores the interaction between Olaparib (OLA), an anticancer drug, and human serum albumin (HSA) on a glassy carbon electrode using cyclic voltammetry and differential pulse voltammetry methods. The study investigates the alterations in the electrochemical behavior of [Fe(CN)6]3–/4– redox pairs in a physiological pH buffer solution due to the OLA-HSA interaction. The electrochemical and kinetic parameters of the redox probe were calculated in the presence of both the drug and protein. Notable variations in these parameters were observed, which can be attributed to the formation of an electro-inactive complex between the protein and drug. The study determined the parameters describing the OLA-HSA complex, including the binding constant and complex stoichiometry. The results revealed the formation of a strong singular drug-albumin complex with a binding constant of 1.12 × 105 M–1. Molecular docking studies supported the experimental findings and provided insights into the binding interactions of OLA with HSA. This study provides valuable insights for future research aimed at enhancing drug delivery systems.

Evaluation of the efficacy of marine natural products against PARP-1/2 proteins in high-grade serous ovarian cancer: insights into MD and SMD simulations

High-grade serous ovarian cancer (HGSOC) is the most malignant and ubiquitous phenotype of epithelial ovarian cancer. Originating in the fallopian tubes and rapidly spreading to the ovaries, this highly heterogeneous disease is a result of serous tubal intraepithelial carcinoma. The proteins known as poly(ADP-ribose) polymerase (PARP) aid in the development of HGSOC by repairing the cancer cells that proliferate and spread metastatically. By using molecular docking to screen 1100 marine natural products (MNPs) from different marine environments against PARP-1/2 proteins, prominent PARP inhibitors (PARPi) were identified. Four compounds, alisiaquinone A, alisiaquinone C, ascomindone D and (+)-zampanolide referred to as MNP-1, MNP-2, MNP-3 and MNP-4, respectively, were chosen based on their binding affinity towards PARP-1/2 proteins, and their bioavailability and drug-like qualities were accessed using ADMET analysis. To investigate the structural stability and dynamics of these complexes, molecular dynamics simulations were performed for 200 ns. These results were compared with the complexes of olaparib (OLA), a PARPi that has been approved by the FDA for the treatment of advanced ovarian cancer. We determined that MNP-4 exhibited stronger binding energies with PARP-1/2 proteins than OLA by using MM/PBSA calculations. Hotspot residues from PARP-1 (E883, M890, Y896, D899 and Y907) and PARP-2 (Y449, F450, A451, S457 and Y460) showed strong interactions with the compounds. To comprehend the unbinding mechanism of MNP-4 complexed with PARP-1/2, steered molecular dynamics (SMD) simulations were performed. We concluded from the free energy landscape (FEL) map that PARP-1/2 are well-stabilised when the compound MNP-4 is bound rather than being pulled away from its binding pockets. This finding provides significant evidence regarding PARPi, which could potentially be employed in the therapeutic treatment of HGSOC. Communicated by Ramaswamy H. Sarma

AURKA blockage as a strategy to overcome resistance to olaparib and platinum in high-grade ovarian cancer (HGOC).

e15198 Background: PARP inhibitors (PARPi) as olaparib (OLA) have become the new standard in maintenance treatment in high grade ovarian carcinoma (HGOC). Thus, there is an important need to identify the mechanisms for PARPi resistance and the strategies to overcome them. Our aim is to assess if in vitro inhibition of AURKA, a protein that plays a relevant role in centrosome spindle formation and mitotic regulation, might overcome olaparib resistance mechanisms in HGOC. Methods: Three HGOC cell lines (PEO1, Kuramochi and SKOV3) were selected, and isogenic resistant pairs generated in vitro, by pulsed exposure to OLA (PEO1-R) or by exposure to growing concentrations of OLA (Kuramochi-R and SKOV-R). AURKA expression was assessed by PCR and Western blot (WB). Cells were OLA-treated before and after the downregulation of AURKA expression by a specific small inhibitor RNA (siRNA) and by pharmacologic inhibition with alisertib. Cell viability was assessed by MTT assay and DNA damage by the evaluation of markers by WB. Combination index of the combo OLA+alisertib was assessed by Chou-Talalay method. Results: AURKA expression was lower at gene and protein levels in resistant cell lines compared to parental. Upon AURKA silencing (Si) of parental (OLA-sensitive) cell lines, no differences in the OLA IC50 values between AURKA-Si and control cells were seen. However AURKA-Si of the OLA-resistant cell lines was associated with a decreased in OLA IC50 as follows: 1) In PEO1R control scramble siRNA (sc) was 226 mM vs SI 128 mM, p < 0.0001; 2) In Kuramochi-R scRNA 268.4 mM vs si 130.2 mM p < 0.0001 and 3) in SKOV3-R scRNA 187 mM vs si 74.7 mM, p < 0.0001 suggesting a role of AURKA-si in overcoming OLA-resistance. Pharmacologic AURKA targeting with the inhibitor alisertib in all 3 sensitive and resistant cell lines showed an increase of DNA damage measured by the expression of H2AX protein by WB. Combination index of the OLA+alisertib in all resistant cell lines showed a synergistic effect of the combination with PEO1-R FA50 = 0.25 (FA25-75 0.2-0.31), Kuramochi-R FA50 = 0.29 (0.19-9.36) and SKOV3-R FA50 = 0.24 (0.19-0.36). Conclusions: AURKA targeting either by silencing or pharmacologic inhibition increased OLA-induced apoptosis or had a synergistic effect in OLA-resistant cell lines suggesting AURKA as a potential target for overcoming OLA resistance.

First-in-human phase I trial of the oral first-in-class ubiquitin specific peptidase 1 (USP1) inhibitor KSQ-4279 (KSQi), given as single agent (SA) and in combination with olaparib (OLA) or carboplatin (CARBO) in patients (pts) with advanced solid tumors, enriched for deleterious homologous recombination repair (HRR) mutations.

3005 Background: USP1 is a deubiquitinase that regulates DNA damage response pathways, such as Translesion Synthesis and Fanconi Anemia pathways. KSQi is a potent, selective small molecule inhibitor of USP1 with anti-proliferative activity in tumors with HRR mutations. The combination of KSQi and PARP inhibitors (PARPi) showed strong synergy in Ovarian and TNBC PDX models, supporting this clinical trial. Methods: This is a 2-part study: Part 1 dose escalation using a BOIN design explored safety, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary anti-tumor activity of KSQi as SA (Arm 1) and in combination with OLA at 200 mg BID (Arm 2) or CARBO at AUC 4 (Arm 3) to determine the MTD and/or recommended dose for expansion (RDE). Part 2 will assess efficacy and safety of the combinations in expansion cohorts. Results: As of Jan 4, 2024, 64 pts (19m/45f; median age 63 y) received KSQi 100 - 1250 mg; Arm 1: 100 mg (3 pts), 150 (3), 200 (4), 300 (6), 450 (5), 650 (9), 900 (7) and 1250 (5); Arm 2: 200 (5), 450 (5) and 900 (5); Arm 3: 200 (3) and 450 (4). Median number of prior therapies was 5, 4 and 3 for Arm 1, 2 and 3, respectively. 29% had prior PARPi in Arm 1, 53% in Arm 2, and 71% in Arm 3. All pts in Arms 2 and 3 had tumors with deleterious HRR mutations. Most common treatment-emergent adverse events (TEAE) were anemia (36%), increased creatinine (33%) as SA, and anemia in combination with OLA (87%) and CARBO (71%). The most common G3+ TEAEs were hyponatremia (12%) as SA and anemia in combination with OLA (73%) and CARBO (29%). DLTs (n = 3) were G3 maculopapular rash at 1250 mg (Arm 1) and G3 WBC decrease at 200 mg, G3 anemia at 450 mg (Arm 2). 30% (19/64) of pts had an interruption in KSQi dosing and 1 (1.6%) discontinued treatment. PK AUC and Cmax increased almost dose-proportionally up to 650 mg. A preliminary review of OLA concentrations following co-administration with KSQi showed no significant impact of OLA at C2D1. Ubiquitinated PCNA induction was observed in paired tumor biopsies from pts receiving KSQi, supporting intra-tumoral USP1 inhibition. A RECIST PR was observed in a fallopian tube cancer pt lasting 7 weeks and SD in 9 pts treated with SA KSQi; best response in combination with OLA was SD in 6 pts and 2 SD with CARBO. Disease control rate at 16 weeks was 28% (Arm 1), 40% (Arm 2) and 29% (Arm 3). Conclusions: KSQi is a first-in-class USP1 inhibitor with an acceptable safety profile as SA and in combination. An MTD was not reached in any Arms. PD results support the mechanism of action of USP1 inhibition. The RDE will be determined in additional back-fill cohorts. Clinical trial information: NCT05240898 .

Exploring the interaction of a potent anti-cancer drug Selumetinib with bovine serum albumin: Spectral and computational attributes.

The binding of drugs to plasma proteins determines its fate within the physiological system, hence profound understanding of its interaction within the bloodstream is important to understand its pharmacodynamics and pharmacokinetics and thereby its therapeutic potential. In this regard, our work delineates the mechanism of interaction of Selumetinib (SEL), a potent anti-cancer drug showing excellent effect against multiple solid tumors, with plasma protein bovine serum albumin (BSA), using methods such as absorption, steady-state fluorescence, time-resolved, fluorescence resonance energy transfer, Fourier transform infrared spectra (FTIR), circular dichroism (CD), synchronous and 3D-fluorescence, salt fluorescence, molecular docking and molecular dynamic simulations. The BSA fluorescence intensity was quenched with increasing concentration of SEL which indicates interactions of SEL with BSA. Stern-Volmer quenching analysis and lifetime studies indicate the involvement of dynamic quenching. However, some contributions from the static quenching mechanism could not be ruled out unambiguously. The association constant was found to be 5.34 × 105 M-1 and it has a single binding site. The Förster distance (r) indicated probable energy transmission between the BSA and SEL. The positive entropy changes and enthalpy change indicate that the main interacting forces are hydrophobic forces, also evidenced by the results of molecular modeling studies. Conformation change in protein framework was revealed from FTIR, synchronous and 3D fluorescence and CD studies. Competitive binding experiments as well as docking studies suggest that SEL attaches itself to site I (subdomain IIA) of BSA where warfarin binds. Molecular dynamic simulations indicate the stability of the SEL-BSA complex. The association energy between BSA and SEL is affected in the presence of different metals differently.

Enhanced cisplatin chemotherapy sensitivity by self-assembled nanoparticles with Olaparib.

Cisplatin (CDDP) is widely used as one kind of chemotherapy drugs in cancer treatment. It functions by interacting with DNA, leading to the DNA damage and subsequent cellular apoptosis. However, the presence of intracellular PARP1 diminishes the anticancer efficacy of CDDP by repairing DNA strands. Olaparib (OLA), a PARP inhibitor, enhances the accumulation of DNA damage by inhibiting its repair. Therefore, the combination of these two drugs enhances the sensitivity of CDDP chemotherapy, leading to improved therapeutic outcomes. Nevertheless, both drugs suffer from poor water solubility and limited tumor targeting capabilities. To address this challenge, we proposed the self-assembly of two drugs, CDDP and OLA, through hydrogen bonding to form stable and uniform nanoparticles. Self-assembled nanoparticles efficiently target tumor cells and selectively release CDDP and OLA within the acidic tumor microenvironment, capitalizing on their respective mechanisms of action for improved anticancer therapy. In vitro studies demonstrated that the CDDP-OLA NPs are significantly more effective than CDDP/OLA mixture and CDDP at penetrating cancer cells and suppressing their growth. In vivo studies revealed that the nanoparticles specifically accumulated at the tumor site and enhanced the therapeutic efficacy without obvious adverse effects. This approach holds great potential for enhancing the drugs' water solubility, tumor targeting, bioavailability, and synergistic anticancer effects while minimizing its toxic side effects.

Combination of Resveratrol and PARP inhibitor Olaparib efficiently deregulates homologous recombination repair pathway in breast cancer cells through inhibition of TIP60-mediated chromatin relaxation.

Recently, we reported that a combination of a natural, bioactive compound Resveratrol (RES) and a PARP inhibitor Olaparib (OLA) deregulated the homologous recombination (HR) pathway, and enhanced apoptosis in BRCA1-wild-type, HR-proficient breast cancer cells. Upon DNA damage, chromatin relaxation takes place, which allows the DNA repair proteins to access the DNA lesion. But whether chromatin remodeling has any role in RES + OLA-mediated HR inhibition is not known. By using in vitro and ex vivo model systems of breast cancer, we have investigated whether RES + OLA inhibits chromatin relaxation and thereby blocks the HR pathway. It was found that RES + OLA inhibited PARP1 activity, terminated PARP1-BRCA1 interaction, and deregulated the HR pathway only in the chromatin fraction of MCF-7 cells. DR-GFP reporter plasmid-based HR assay demonstrated marked reduction in HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. RES + OLA efficiently trapped PARP1 at the DNA damage site in the chromatin of MCF-7 cells. Unaltered expressions of HR proteins were found in the chromatin of PARP1-silenced MCF-7 cells, which confirmed that RES + OLA-mediated DNA damage response was PARP1-dependent. Histone Acetyltransferase (HAT) activity and histone H4 acetylation assays showed reduction in HAT activity and H4 acetylation in RES + OLA-treated chromatin fraction of cells. Western blot analysis showed that the HAT enzyme TIP60, P400 and acetylated H4 were downregulated after RES + OLA exposure. In the co-immunoprecipitation assay, it was observed that RES + OLA caused abolition of PARP1-TIP60-BRCA1 interaction, which suggested the PARP1-dependent TIP60-BRCA1 association. Unaltered expressions of PAR, BRCA1, P400, and acetylated H4 in the chromatin of TIP60-silenced MCF-7 cells further confirmed the role of TIP60 in PARP1-mediated HR activation in the chromatin. Similar results were obtained in ex vivo patient-derived primary breast cancer cells. Thus, the present study revealed that RES + OLA treatment inhibited PARP1 activity in the chromatin, and blocked TIP60-mediated chromatin relaxation, which, in turn, affected PARP1-dependent TIP60-BRCA1 association, resulting in deregulation of HR pathway in breast cancer cells.

Evaluation of Anticancer Efficacy of D-α-Tocopheryl Polyethylene-Glycol Succinate and Soluplus® Mixed Micelles Loaded with Olaparib and Rapamycin Against Ovarian Cancer.

Ovarian cancer has the highest mortality rate and lowest survival rate among female reproductive system malignancies. There are treatment options of surgery and chemotherapy, but both are limited. In this study, we developed and evaluated micelles composed of D-α-tocopheryl polyethylene-glycol (PEG) 1000 succinate (TPGS) and Soluplus® (SOL) loaded with olaparib (OLA), a poly(ADP-ribose)polymerase (PARP) inhibitor, and rapamycin (RAPA), a mammalian target of rapamycin (mTOR) inhibitor in ovarian cancer. We prepared micelles containing different molar ratios of OLA and RAPA embedded in different weight ratios of TPGS and SOL (OLA/RAPA-TPGS/SOL) were prepared and physicochemical characterized. Furthermore, we performed in vitro cytotoxicity experiments of OLA, RAPA, and OLA/RAPA-TPGS/SOL. In vivo toxicity and antitumor efficacy assays were also performed to assess the efficacy of the mixed micellar system. OLA/RAPA-TPGS/SOL containing a 4:1 TPGS:SOL weight ratio and a 2:3 OLA:RAPA molar ratio showed synergistic effects and were optimized. The drug encapsulation efficiency of this formulation was >65%, and the physicochemical properties were sustained for 180 days. Moreover, the formulation had a high cell uptake rate and significantly inhibited cell migration (**p < 0.01). In the in vivo toxicity test, no toxicity was observed, with the exception of the high dose group. Furthermore, OLA/RAPA-TPGS/SOL markedly inhibited tumor spheroid and tumor growth in vivo. Compared to the control, OLA/RAPA-TPGS/SOL showed significant tumor inhibition. These findings lay a foundation for the use of TPGS/SOL mixed micelles loaded with OLA and RAPA in the treatment of ovarian cancer.

Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer.

In our previous research, we proved that ailanthone (AIL) inhibits the growth of gastric cancer (GC) cells and causes apoptosis by inhibiting P23. However, we still find some GC organoids are insensitive to AIL. We have done some sequencing analysis and found that the insensitive strains are highly expressed in PARP1. In this study, we investigated whether AIL can enhance the anti-tumour effect of PARPi in GC. CCK8 and spheroid colony formation assay were used to measure anti-tumour effects. SynergyFinder software was used to calculate the synergy score of the drug combination and flow cytometry was used to detect apoptosis. Western blot, IHC, IF tests were used to measure protein expression. Finally, nude mouse xenograft models were used to verify the invitro mechanisms. High expression of PARP1 was found to be the cause of drug insensitivity. When AIL is paired with a PARP1 inhibitor, olaparib (OLP), drug sensitivity improves. We discovered that this combination functions by blocking off HSP90-BRCA1 interaction and inhibiting the activity of PARP1, thus in turn inhibiting the homologous recombination deficiency and base excision repair pathway to finally achieve synthetic lethality through increased sensitivity. Moreover, P23 can regulate BRCA1 in GC invitro. This study proves that the inhibitory effect of AIL on BRCA1 allowed even cancer cells with normal BRCA1 function to be sensitive to PARP inhibitors when it is simultaneously administered with OLP. The results greatly expanded the scope of the application of PARPi.

Delineation of prototypical degradation mechanism, characterization of unknown degradation impurities by liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry and stability-indicating analytical method of selumetinib.

Selumetinib (SELU) was recently approved by the US Food and Drug Administration (US FDA) in 2020. However, the degradation impurities of SELU have not been characterized or identified to date. The mechanism for impurity formation and the degradation behavior have not been previously studied. This study aims to elucidate the prototypical degradation mechanism of SELU. Furthermore, the degradation impurities have been identified using LC-quadrupole-time-of-flight tandem mass spectrometry and are reported in this article for the first time. In addition, a stability-indicating analytical method (SIAM) has been developed for this drug. Forced degradation studies revealed the degradation of SELU under various stress conditions, including hydrolytic stress (acid and base), oxidative stress, and photolytic stress (ultraviolet and visible). Three degradation impurities were identified. This article presents the first validated SIAM, capable of accurately quantifying SELU in the presence of its degradation impurities. Furthermore, we have proposed the degradation pathway for SELU and its degradation impurities, a first in the field. The developed SIAM can find applications in process development and quality assurance of SELU in both research laboratories and pharmaceutical industries. Moreover, the identified degradation impurities may serve as impurity standards for quality control testing in pharmaceutical industries.

Improving the physicochemical and pharmacokinetic properties of olaparib through cocrystallization strategy

Olaparib (OLA) is the first PARP inhibitor worldwide used for the treatment of ovarian cancer. However, the oral absorption of OLA is extremely limited by its poor solubility. Herein, pharmaceutical cocrystallization strategy was employed to optimize the physicochemical and pharmacokinetic properties. Four cocrystals of OLA with oxalic acid (OLA-OA), malonic acid (OLA-MA), fumaric acid (OLA-FA) and maleic acid (OLA-MLA) were successfully discovered and characterized. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirmed the formation of cocrystals rather than salts, and the possible hydrogen bonding patterns were analyzed through molecular surface electrostatic potential calculations. The in vitro and in vivo evaluations indicate that all of the cocrystals demonstrate significantly improved dissolution performance, oral absorption and tabletability compared to pure OLA. Among them, OLA-FA exhibit sufficient stability and the most increased Cmax and AUC0-24h values that were 11.6 and 6.1 times of free OLA, respectively, which has great potential to be developed into the improved solid preparations of OLA.

134P Doxorubicin and olaparib (OLA) synergism in high-grade serous ovarian (HGOC) and triple-negative breast cancer (TNBC) cell lines with olaparib-resistance

134P Doxorubicin and olaparib (OLA) synergism in high-grade serous ovarian (HGOC) and triple-negative breast cancer (TNBC) cell lines with olaparib-resistance

Physiologically-based pharmacokinetic modeling for optimal dosage prediction of olaparib when co-administered with CYP3A4 modulators and in patients with hepatic/renal impairment

This study aimed to develop a physiologically-based pharmacokinetic (PBPK) model to predict the maximum plasma concentration (Cmax) and trough concentration (Ctrough) at steady-state of olaparib (OLA) in Caucasian, Japanese and Chinese. Furthermore, the PBPK model was combined with mean and 95% confidence interval to predict optimal dosing regimens of OLA when co-administered with CYP3A4 modulators and administered to patients with hepatic/renal impairment. The dosing regimens were determined based on safety and efficacy PK threshold Cmax (< 12,500 ng/mL) and Ctrough (772–2500 ng/mL). The population PBPK model for OLA was successfully developed and validated, demonstrating good consistency with clinically observed data. The ratios of predicted to observed values for Cmax and Ctrough fell within the range of 0.5 to 2.0. When OLA was co-administered with a strong or moderate CYP3A4 inhibitor, the recommended dosing regimens should be reduced to 100 mg BID and 150 mg BID, respectively. Additionally, the PBPK model also suggested that OLA could be not recommended with a strong or moderate CYP3A4 inducer. For patients with moderate hepatic and renal impairment, the dosing regimens of OLA were recommended to be reduced to 200 mg BID and 150 mg BID, respectively. In cases of severe hepatic and renal impairment, the PBPK model suggested a dosing regimen of 100 mg BID for OLA. Overall, this present PBPK model can determine the optimal dosing regimens for various clinical scenarios involving OLA.

Development and Comparative Evaluation of Two Different Label-Free and Sensitive Fluorescence Platforms for Analysis of Olaparib: A Recently FDA-Approved Drug for the Treatment of Ovarian and Breast Cancer.

Olaparib (OLA) is a PARP inhibitor drug which has been recently approved by the Food and Drug Administration (FDA) for the treatment of ovarian and breast cancer. A convenient analytical tool for the quantitation of OLA in its dosage form and plasma samples was urgently needed. This study describes, for the first time, the development of two different label-free and sensitive fluorescence-based platforms for the pharmaceutical and bioanalysis of OLA. These platforms were microwell-assisted with a fluorescence microplate reader (MW-FLR) and high-performance liquid chromatography with fluorescence detection (HPLC-FD). Both MW-FLR and HPLC-FD employed the native fluorescence of OLA as an analytical signal. The MW-FLR involved measuring the fluorescence signals in 96-well white-opaque plates. The HPLC-FD involved chromatographic separation of OLA and duvelisib (DUV), as an internal standard on a Nucleosil-CN HPLC column (250 mm length × 4.6 mm i.d., 5 µm particle diameter) with a mobile phase composed of acetonitrile: water (25:75, v/v) pumped at a flow rate of 1.7 mL/min. Elution of OLA and DUV was detected using a fluorescence detector. The optimal conditions of both MW-FLR and HPLC-FD were established, and they were validated according to the guidelines of the International Council for Harmonization for the validation of analytical procedures. The linear ranges of MW-FLR and HPLC-FD were 25-1000 and 5-200 ng/mL, respectively, with limits of detection of 15 and 1.7 ng/mL, respectively. The accuracy and precision of both platforms were confirmed as the recovery values were ≥98.2% and the relative standard deviations (RSD) were ≤2.89%. Both methodologies were satisfactorily applied to the quantitation of OLA in its commercial dosage form (Lynparza® tablets) and plasma samples with high accuracy and precision. The greenness of both MW-FLR and HPLC-FD was assessed using two different multiple parameter-based metric tools, and the results proved their greenness and adherence to the requirements of green analytical approaches. Both platforms have simple procedures and acceptable levels of analytical throughput. In conclusion, the proposed MW-FLR and HPLC-FD are valuable tools for routine use in quality control and clinical laboratories for the quantitation of OLA for the purposes of pharmaceutical quality control, pharmacokinetic studies, and bioequivalence testing.

A Comparative Study of Molecular Imprinting Techniques Used for Fabrication of Electrochemical Sensor of Olaparib

This study reported the molecular imprinting process investigated by two techniques: photopolymerization (PP) and thermal polymerization (TP). These techniques were integrated with an electrochemical sensor to determine the new generation anticancer drug Olaparib (OLP), which is a poly(ADP-ribose) polymerase (PARP) inhibitor with high sensitivity and selectivity. The PP-MIP film was formed by PP, while TP-MIP film was formed by TP on a glassy carbon electrode (GCE). In the PP-MIP(OLP)/GCE sensor, which was developed using PP, 4-aminobenzoic acid (4-AB) as a functional monomer was designed, and obtained in the presence of basic monomer (HEMA, 2-hydroxyethyl methacrylate), crosslinker (EGDMA, ethylene glycol dimethacrylate), and initiator (2-hydroxy-2-methyl propiophenone) by keeping it under a UV lamp at 365 nm. For TP-MIP(OLP)/GCE was obtained by using a mixture of 4-AB as a functional monomer, NH3 solution, surfactant sodium dodecyl sulfate (SDS), and tetraethyl orthosilicate (TEOS): ethanol (1:1, v/v) and keeping it in an oven at 50 °C for 40 min. After each process, the GCE surfaces were characterized via scanning electron microscope and electrochemical techniques (cyclic voltammetry and electrochemical impedance spectroscopy). The required optimization studies were performed for both techniques. After obtaining the optimum MIP-integrated electrochemical sensors, the analytical performances of PP-MIP(OLP)/GCE and TP-MIP(OLP)/GCE were evaluated, and the validation parameters were compared. The linear ranges of PP-MIP(OLP)/GCE and TP-MIP(OLP)/GCE are 0.1–1 nM. The PP-MIP(OLP)/GCE and TP-MIP(OLP)/GCE were applied to commercial serum samples and pharmaceutical dosage forms. The selectivity of proposed sensors was also proven using similar structures of OLP through imprinting factor. The results show that the proposed PP-MIP(OLP)/GCE and TP-MIP(OLP)/GCE sensors have good selectivity, high sensitivity, accuracy, and precision. The determination of OLP was achieved with these electrochemical sensors for the first time.

AXL pathway as a target to overcome resistance to PARP inhibitors in high-grade serous ovarian (HGSOC) and triple-negative breast cancer (TNBC) cell lines.

e17502 Background: PARP inhibitors (PARPi) as olaparib (OLA) have become the new standard in maintenance treatment in high grade serous ovarian carcinoma (HGSOC) and triple negative breast cancer (TNBC). Thus there is an important need to identify the mechanisms for PARPi resistance and the strategies to overcome it. Our aim is to intervene in vitro AXL, a receptor tyrosine kinase, linking its function to the OLA-resistance mechanism and assess its suitability as a therapeutical target to overcome PARPi-resistance. Methods: Three cell lines, 2 from HGSOC (PEO1-S and Kuramochi-S) and 1 from TNBC (MDA-MB-231-S) were selected, isogenic resistant pairs generated in vitro by pulsed exposure to OLA (PEO1-R) or by exposure to growing concentrations of OLA (Kuramochi-R and MDA-MB-231-R) were obtained. Resistance was confirm by the comparison of IC50 of OLA in -S vs -R lines. AXL expression was assessed by western blot (WB). Cells were treated with OLA before and after the downregulation of AXL expression by a specific siRNA. Cell viability was assessed by MTT assay and DNA damage by the evaluation of markers by western blot (WB). Results: AXL expression was maintained upon OLA treatment in all 3 resistant cell lines. However, AXL expression was reduced upon OLA exposure in 3 sensible parental cell lines. AXL silencing (Si) lead to a significantly decrease of OLA IC50 in all 3 sensitive and resistant cell lines. For sensitive cell lines, IC50 of OLA was in 1) PEO1S with control (scramble) was 5.79 mM vs 2.24 mM after silencing with siRNA, p &lt; 0.0001; 2) Kuramochi-S scramble OLA IC50 was 481.8 mM vs 199.7 mM with siRNA, p &lt; 0.0001 and 3) MDA-MB-231-S with control of scramble the OLA IC50 was 61.62 mM vs 26.21 mM with siRNA, p = 0.001. For resistant cell lines, 4) PEO1R control with scramble 91.49 mM vs siRNA 45.72 mM, p &lt; 0.0001; 5) Kuramochi-R scramble OLA IC50 579.8 mM vs 219.8 mM with siRNA, p = 0.0008 and 6) 231-R scramble OLA IC50 68.22 mM vs 9.247 mM with siRNA, p &lt; 0.0001. These results suggest that AXL downregulation increases sensitivity to OLA in both sensitive and resistant cell lines. Histone γ-H2AX was analysed pre and post silencing and both increased when treating with OLA, especially when AXL was downregulated in alll three resistant cell lines. Conclusions: This in vitro study suggest a potential role of AXL in OLA resistance and AXL downregulation migh represent a potential mechanism to overcome resistance to OLA in ovarian and TNBC cell lines.

The combination of APR-246 and carboplatin in olaparib-resistant high grade serous ovarian cancer (HGSOC) and triple negative breast cancer (TNBC) cell lines.

e17504 Background: PARP inhibitors (PARPi), as Olaparib (OLA), have changed the therapeutic landscape in HGSOC and TNBC. However, most patients will develop resistance to PARPi. Although resistance mechanisms are poorly understood, it is suggested that BRCA mutations reversions or cell cycle alterations could be involved. APR-246 is a new targeted agent that restores p53 in TP53-mutated cells. The aim of our study was to assess the potential synergism of the combination of carboplatin (carbo) plus APR-246 in both OLA-sensitive and OLA-resistant HGSOC and TNBC cell lines. Methods: We selected two HGSOC (PEO1-S and Kuramochi-S) and one TNBC TP53 mutant cell lines (MDA-MB-231-S). Resistance to OLA was generated by pulse doses with IC75 OLA for 24 hours and recovered for two weeks during a period of 6 months (PEO1-R) and to increased concentrations of OLA for 72 hours during a period of 1 year (Kuramochi-R, MDA-MB-231-R). Next-generation sequencing was performed in order to assess mutations. IC50 of APR-246 and Carbo and the combination of both (combo) were calculated for Kura-S/R, PEO1-S/R and 231-S/R cell lines. Synergism between both drugs was assessed with Chou-Talalay method for combination index (CI) at Fa50. DNA damage was assessed by gamma-H2AX expression by western-blot (WB). Results: 231-S harboured a TP53 mutation (mut) (missense R280K) and BRCA wildtype (wt), in PEO1-S BRCA2 mut p.Y1655* and TP53 mut was detectable. Kuramochi-S was TP53 mut (missense D816Y) and BRCA2 mut. BRCA and p53 mutation remained unchanged in the resistant lines generated.The mutation profile of each line and the IC50 of APR-246, carbo and combo are shown in the table. IC50 of carboplatin is superior in all 3 resistant cell lines when compared to their sensitive parental line, suggesting a potential cross-resistance to platinum in OLA-resistance. The combination of carbo and APR-246 is synergistic even in OLA-resistant cell lines, in fact in those cross-resistant lines values of synergism by Chou-Talalay are improved by the combo suggesting a potential role of this combination in PARPi resistant cell lines. WB showed an increased DNA damage with the combo when compared to either single agent, with an increase in H2AX in all lines either sensitive or resistant. Conclusions: The combination of APR246 and carboplatin is synergistic in all 3 studied cell lines but also in the OLA resistant ones. The combo increased its synergistic effect suggesting a potential role of combo in OLA-resistant tumors. [Table: see text]

Enzalutamide and olaparib synergistically suppress castration-resistant prostate cancer progression by promoting apoptosis through inhibiting nonhomologous end joining pathway.

Recent studies revealed the relationship among homologous recombination repair (HRR), androgen receptor (AR), and poly(adenosine diphosphate-ribose) polymerase (PARP); however, the synergy between anti-androgen enzalutamide (ENZ) and PARP inhibitor olaparib (OLA) remains unclear. Here, we showed that the synergistic effect of ENZ and OLA significantly reduced proliferation and induced apoptosis in AR-positive prostate cancer cell lines. Next-generation sequencing followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed the significant effects of ENZ plus OLA on nonhomologous end joining (NHEJ) and apoptosis pathways. ENZ combined with OLA synergistically inhibited the NHEJ pathway by repressing DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and X-ray repair cross complementing 4 (XRCC4). Moreover, our data showed that ENZ could enhance the response of prostate cancer cells to the combination therapy by reversing the anti-apoptotic effect of OLA through the downregulation of anti-apoptotic gene insulin-like growth factor 1 receptor (IGF1R) and the upregulation of pro-apoptotic gene death-associated protein kinase 1 (DAPK1). Collectively, our results suggested that ENZ combined with OLA can promote prostate cancer cell apoptosis by multiple pathways other than inducing HRR defects, providing evidence for the combined use of ENZ and OLA in prostate cancer regardless of HRR gene mutation status.