Simultaneous estimation of paclitaxel and bortezomib via LC-MS/MS: pharmaceutical and pharmacokinetic applications.

Aim & Objective: This study evaluates the potential of combining paclitaxel (PTX) and bortezomib (BTZ) for breast cancer therapy. Materials & Methods: The nanoformulation was optimized via Box-Behnken Design (BBD), with method validation adhering to US-FDA guidelines. Results: Multiple reaction monitoring transitions for PTX, BTZ and internal standard were m/z 855.80→286.60, 366.80→226.00 and 179.80→110.00, respectively. Elution done on C18 Luna column with 0.1% FA in MeOH:10 mM ammonium acetate. The size of nanoformulation was 133.9 ± 1.97 nm, PDI 0.19 ± 0.01 and zeta potential -19.20 ± 1.36 mV. Pharmacokinetics showed higher Cmax for PTX-BTZ-NE (313.75 ± 10.71 ng/ml PTX, 11.92 ± 0.53 ng/ml BTZ) versus free PTX-BTZ (104 ± 13.06 ng/ml PTX, 1.9 ± 0.08 ng/ml BTZ). Conclusion: Future findings will contribute to the treatment of breast cancer using PTX and BTZ.

[Box: see text].

Simultaneous estimation of rutin and donepezil through RP-HPLC: implication in pharmaceutical and biological samples.

Aim: A HPLC method was developed and validated for the novel combination of rutin (RN) and donepezil (DNP). Materials & methods: RN and DNP were simultaneously eluted through a C18 column (Ø 150 × 4.6 mm) with a 60:40 v/v ratio of 0.1% formic acid aqueous solution to methanol at 0.5 ml/min. Results: The purposed method was found linear, selective, reproducible, accurate and precise with percent RSD less than 2. The limit of quantification for RN and DNP was found 3.66 and 3.25 µg/ml, respectively. Conclusion: Validated as per the ICH guidelines, the developed method efficiently quantified RN and DNP co-loaded in DQAsomes (121 nm) estimating matrix effect, release profile, entrapment efficiency, loading efficiency and in vivo plasma kinetics.

[Box: see text].

Synchronized Codelivery of Combination Chemotherapies Intratumorally Using a Lipidic Lyotropic Liquid Crystal System.

In this work, an injectable in situ depot-forming lipidic lyotropic liquid crystal (L3C) system is developed to codeliver a precisely synchronized combination of chemotherapeutics intratumorally. The developed L3C system is composed of amphiphilic lipids and surfactants, including monoolein, phosphatidylcholine, tocopherol acetate, and d-α-tocopherol polyethylene glycol 1000 succinate. Owing to its amphiphilic nature, the developed formulation can coaccommodate both hydrophobic and hydrophilic chemotherapeutic moieties simultaneously. The study presents a proof of concept by designing a combination chemotherapy regimen in vitro and demonstrating its in vivo translation using doxorubicin and paclitaxel as model hydrophilic and hydrophobic drug moieties, respectively. The synchronized combination of the two chemotherapeutics with maximum synergistic activity was identified, coloaded in the developed L3C system at predefined stoichiometric ratios, and evaluated for antitumor efficacy in the 4T1 breast tumor model in BALB/c mice. The drug-loaded L3C formulation is a low-viscosity injectable fluid with a lamellar phase that transforms into a hexagonal mesophase depot system upon intratumoral injection. The drug-loaded depot system locally provides sustained intratumoral delivery of the chemotherapeutics combination at their precisely synchronized ratio for over a period of one month. Results demonstrate that the exposure of the tumor to the precisely synchronized intratumoral chemotherapeutics combination via the developed L3C system resulted in significantly higher antitumor activity and reduced cardiotoxicity compared to the unsynchronized combination chemotherapy or the synchronized but uncoordinated drug delivery administered by a conventional intravenous route. These findings demonstrate the potential of the developed L3C system for achieving synchronized codelivery of the chemotherapeutics combination intratumorally and improving the efficacy of combination chemotherapy.

Development and approval of novel injectables: enhancing therapeutic innovations.

INTRODUCTION: Novel injectables possess applications in both local and systemic therapeutics delivery. The advancement in utilized materials for the construction of complex injectables has tremendously upgraded their safety and efficacy. AREAS COVERED: This review focuses on various strategies to produce novel injectables, including oily dispersions, in situ forming implants, injectable suspensions, microspheres, liposomes, and antibody-drug conjugates. We herein present a detailed description of complex injectable technologies and their related drug formulations permitted for clinical use by the United States Food and Drug Administration (USFDA). The excipients used, their purpose and the challenges faced during manufacturing such formulations have been critically discussed. EXPERT OPINION: Novel injectables can deliver therapeutic agents in a controlled way at the desired site. However, several challenges persist with respect to their genericization. Astronomical costs incurred by innovator companies during product development, complexity of the product itself, supply limitations with respect to raw materials, intricate manufacturing processes, patent evergreening, product life-cycle extensions, relatively few and protracted generic approvals contribute to the exorbitant prices and access crunch. Moreover, regulatory guidance are grossly underdeveloped and significant efforts have to be directed toward development of effective characterization techniques.

Quantification of Arbortristoside-A isolated from Nyctanthes arbor-tristis using HPLC: Method development and pharmaceutical applications.

Arbortristoside-A (Arbor-A) is a naturally occurring iridoid glycoside and herbal-based lead molecule with proven medicinal potential. Aiming at the development of an efficient analytical tool for the quantification of Arbor-A in pharmaceutical dosage forms, in the presented work, we developed an economical, fast, and sensitive RP-HPLC-UV method and validated the procedure as per the ICH guidelines, Q2(R1). The chromatographic separation was accomplished under the optimised experimental conditions using an HPLC system with an LC-2010 autosampler, a PDA detector, and a Phenomenex C18 column with the mobile phase composed of a 70:30 (v/v) water-acetonitrile mixture eluting isocratically at a flow rate of 1 mL/min at ambient temperature, and UV detection at 310 nm. Arbor-A showed a sharp peak at the retention time of 5.60 min and exhibited linearity (R2 = 0.9988) with LOD and LOQ of 0.50 µg/mL and 1.50 µg/mL, respectively. The accuracy of the method was 98.33-101.36 % with acceptable intra-day and inter-day precisions as well as robustness (<2% RSD). To ratify the applicability of the presented approach in emerging pharmaceuticals, a nanoformulation loaded with Arbor-A was designed and analysed utilising the provided methodology. The method has also enabled to determine the degradation kinetics of Arbor-A under stress conditions, etcetera, employing forced degradation and short term stability studies.

Technology Transfer of a Validated RP-HPLC Method for the Simultaneous Estimation of Andrographolide and Paclitaxel in Application to Pharmaceutical Nanoformulation.

Many analytical methods are reported for simultaneous estimation of pharmaceutical dosages form. However, only a few are reproducible at an industrial scale. The proposed research aims to establish a technology transfer (TT) protocol between two laboratories (Lab-X, originator) with binary and (Lab-Y, receiver) with quaternary high-performance liquid chromatography (HPLC) system. Thus, utilizing reverse-phase HPLC (RP-HPLC), a robust, sensitive and repeatable analytical method has been developed, validated and TT between two laboratories for simultaneous estimation of Andrographolide (AG) and Paclitaxel (PTX). The method has been developed on a Phenomenex Luna C18 column (150 x 4.6, 5) sustained at 40°C and validated under the International Conference on Harmonisation (ICH) Q2 (R1) regulatory guideline and TT USP chapter 1224. The mobile phase consisted of MilliQ (pH = 3) and a combination of acetonitrile and methanol (1:1) in the ratio 50:50 with a flow rate of 0.45 mL/min, linear gradient elution in both labs. The AG and PTX were detected on the PDA detector at 224 and 227 nm wavelength with retention time of 4.5 ± 0.34 and 8.2 ± 0.02 min and limit of detection was found 0.028 ± 0.004 µg/mL, and 0.028 ± 0.0007 µg/mL, whereas limit of quantification as 0.086 ± 0.011 µg/mL and 0.088 ± 0.0014 µg/mL respectively in both labs. Throughout, this approach we have proved that proposed method is repeatable in both labs, and it can be used to quantify AG and PTX in developed pharmaceutical nano-formulations.

Ratiometric codelivery of Paclitaxel and Baicalein loaded nanoemulsion for enhancement of breast cancer treatment.

The most prevalent clinical option for treating cancer is combination chemotherapy. In combination therapy, assessment and optimization for obtaining a synergistic ratio could be obtained by various preclinical setups. Currently, in vitro optimization is used to get synergistic cytotoxicity while constructing combinations. Herein, we co-encapsulated Paclitaxel (PTX) and Baicalein (BCLN) with TPP-TPGS1000 containing nanoemulsion (TPP-TPGS1000-PTX-BCLN-NE) for breast cancer treatment. The assessment of cytotoxicity of PTX and BCLN at different molar weight ratios provided an optimized synergistic ratio (1:5). Quality by Design (QbD) approach was later applied for the optimization as well as characterization of nanoformulation for its droplet size, zeta potential and drug content. TPP-TPGS1000-PTX-BCLN-NE significantly enhanced cellular ROS, cell cycle arrest, and depolarization of mitochondrial membrane potential in the 4T1 breast cancer cell line compared to other treatments. In the syngeneic 4T1 BALB/c tumor model, TPP-TPGS1000-PTX-BCLN-NE outperformed other nanoformulation treatments. The pharmacokinetic, biodistribution and live imaging studies pivoted TPP-TPGS1000-PTX-BCLN-NE enhanced bioavailability and PTX accumulation at tumor site. Later, histology studies confirmed nanoemulsion non-toxicity, expressing new opportunities and potential to treat breast cancer. These results suggested that current nanoformulation can be a potential therapeutic approach to effectively address breast cancer therapy.

Enhanced apoptosis and mitochondrial cell death by paclitaxel-loaded TPP-TPGS1000-functionalized nanoemulsion.

Background: The present research was designed to develop a nanoemulsion (NE) of triphenylphosphine-D-α-tocopheryl-polyethylene glycol succinate (TPP-TPGS1000) and paclitaxel (PTX) to effectively deliver PTX to improve breast cancer therapy. Materials & methods: A quality-by-design approach was applied for optimization and in vitro and in vivo characterization were performed. Results: The TPP-TPGS1000-PTX-NE enhanced cellular uptake, mitochondrial membrane depolarization and G2M cell cycle arrest compared with free-PTX treatment. In addition, pharmacokinetics, biodistribution and in vivo live imaging studies in tumor-bearing mice showed that TPP-TPGS1000-PTX-NE had superior performance compared with free-PTX treatment. Histological and survival investigations ascertained the nontoxicity of the nanoformulation, suggesting new opportunities and potential to treat breast cancer. Conclusion: TPP-TPGS1000-PTX-NE improved the efficacy of breast cancer treatment by enhancing its effectiveness and decreasing drug toxicity.

Combining LC-MS/MS profiles with network pharmacology to predict molecular mechanisms of the hyperlipidemic activity of Lagenaria siceraria stand.

ETHNOPHARMACOLOGICAL RELEVANCE: Lagenaria siceraria Stand. (Family: Cucurbitaceae), popularly known as bottle gourd, is traditionally used in Ayurvedic medicine as a food plant, especially in hypertension and obesity. AIM OF THE STUDY: Investigations were undertaken to assign novel lead combinations from this common food plant to multi-molecular modes of actions in the complex disease networks of obesity and hypertension. LC-MS/MS based metabolite screening, in-vivo high fat diet induced hyperlipidemia animal study and network pharmacology explorations of the mechanism of action for lipid lowering effects including a neighbourhood community approach for molecular combinations were performed. MATERIAL AND METHODS: Major chemical constituents of the fruits of LS (LSFE) were analysed by HPLC-DAD-MS/MS-QTOF. Wistar albino rats (n = 36), divided into 6 groups (n = 6) received either no treatment or a high-fat diet along with LSFE or Atorvastatin. Lipid profiles and biochemical parameters were evaluated. In silico cross-validated network analyses using different databases and Cytospace were applied. RESULTS: Profiling of LSFE revealed 18 major constituents: phenolic acids like p-Coumaric acid and Ferulic acid, the monolignolconferyl alcohol, the flavonoid glycosides hesperidin and apigenin-7-glucoside. Hyperlipidemic animals treated with LSFE (200 mg/kg, 400 mg/kg, 600 mg/kg) showed a significant improvement of their lipid profiles after 30 days of treatment. Network pharmacology analyses for the major 18 compounds revealed enrichment of the insulin and the ErbB signalling pathway. Novel target node combinations (e.g. AKR1C1, AGXT) including their connection to different pathways were identified in silico. CONCLUSIONS: The combined in vivo and bioinformatics analyses propose that lead compounds of LSFE act in combination on relevant targets of hyperlipidemia. Perturbations of the IRS→Akt→Foxo1 cascade are predicted which suggest further clinical investigation towards development of safe natural alternative to manage hyperlipidemia.

HPLC method for simultaneous estimation of paclitaxel and baicalein: pharmaceutical and pharmacokinetic applications.

Aim: A novel HPLC method was developed and validated for the simultaneous estimation of paclitaxel (PTX) and baicalein (BAC). Materials & methods: The analytes were resolved in a C18 column using the aqueous solution of formic acid (0.10% v/v) and MeOH (30:70 v/v). Results: The developed method was found to be linear over the concentration ranges 0.039-10 µg/ml and 0.019-10 µg/ml for PTX and BAC, respectively. The lower limits of quantification obtained were 0.042 µg/ml and 0.361 µg/ml for PTX and BAC, respectively. Conclusion: The developed method was found to be precise and accurate as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines, for simultaneous estimation of PTX and BAC, having an application in formulation development and bioanalytical studies.

Validated HPLC-UV Method for Simultaneous Estimation of Paclitaxel and Doxorubicin Employing Ion Pair Chromatography: Application in Formulation Development and Pharmacokinetic Studies.

Though paclitaxel (PTX) and doxorubicin (DOX) are amongst the most widely used and investigated drug pair for combination chemotherapy but surprisingly, not a single validated HPLC-UV method is available to analyze PTX and DOX simultaneously. So, herein a HPLC-UV method is developed and validated for the same, filling an indispensable gap in the literature. As these two moieties have characteristically different polarities, resolving them under the common chromatographic conditions is a challenging task. Herein, the principle of ion pair chromatography is utilized to resolve these two moieties on a C18 column employing an isocratic mobile phase comprised of acetonitrile and octane sulfonic acid buffer (67 : 37) and detected simultaneously at 231 nm using a UV detector only. The retention time is 4.4 and 7.2 min for PTX and DOX, respectively, with a total analysis time of less than 10 minutes, suitable for the formulation development and research, while LOQ is less than 0.066 µg/ml for both the drugs, suitable for the therapeutic drug monitoring at preclinical and clinical research setup. To substantiate the applicability of the developed method, a nanoformulation coloaded with PTX and DOX was designed and analyzed using the developed protocol. The method is also applied successfully to study the plasma kinetic profile of both the moieties simultaneously in Balb/c mice. Further, the method is validated as per the ICH guidelines fulfilling the unmet need of a validated analytical tool to simultaneously estimate PTX and DOX. Moreover, the results suggest that the principal of common ion chromatography demonstrated here can also be applied further for the simultaneous chromatographic separation of other polar and nonpolar moieties too. Consequently, the reported method surely will advance the toolset required for the precision-based combination chemotherapy.

Development of asolectin-based liposomal formulation for controlled and targeted delivery of erlotinib as a model drug for EGFR monotherapy.

The present investigation was envisaged to develop liposomal formulation for efficacious and targeted delivery of epidermal growth factor receptor (EGFR) inhibitor (erlotinib) against pancreatic cancer. The marketed formulations bearing current EGFR inhibitors exhibit serious adverse effects including severe skin, hemolytic and gastrointestinal toxicity. To address the obstacles, we have developed the liposomal formulation, by ether injection method, comprising erlotinib, a tyrosine kinase EGFR inhibitor, proposed to be targeted through enhanced permeability and retention effect (EPR) effect against pancreatic cancer. On succeeding, the liposomes were characterized for various pharmaceutical attributes. The developed liposomes found to sustain a particle size of 121 ± 10.7 nm, whereas PDI of 0.22 ± 0.01 with the surface charge value of -33.7 ± 2.30 mV. The entrapment efficiency and drug loading were found to be 82.60 and 15.89 (%w/w), respectively. The hemolysis study suggested that the developed formulation was safer compared with native drug solution. The proof of concept for enhanced efficacy and decreased toxicity has been established through in vitro assays. The IC50 for free erlotinib and formulation was found to be 2.0 ± 0.3 µg/ml and 1.1 ± 0.1 µg/ml, respectively. The effectivity was evident by cellular uptake study and apoptosis, whereas cell cycle arrest study indicated that erlotinib arrests the G0/G1 phase of cell cycle. Further the erlotinib-asolectin liposomal formulation enhanced cytotoxicity in PANC-1 cells at relatively low dose, proving to be an alternative for current chemotherapeutics against pancreatic cancer.

UPLC-QTOF-MS analysis of a carbonic anhydrase-inhibiting extract and fractions of Luffa acutangula (L.) Roxb (ridge gourd).

INTRODUCTION: Luffa acutangula (L.) Roxb, commonly known as ridge gourd (cucurbitaceae), is a common vegetable cultivated in India. It is also a well-used medicinal plant in Indian traditional medicine. OBJECTIVES: To analyse the phenolics content of the most potent carbonic anhydrase-inhibiting fraction from an extract of L. acutangula. MATERIALS AND METHODS: An aqueous ethanol extract of dried fruits of L. acutangula was successively fractionated into petroleum ether, dichloromethane and ethyl acetate. The extract and subsequent fractions were assessed for carbonic anhydrase-inhibitory activity and the enzyme inhibition kinetics were determined for the most active fraction. Total phenolic and flavonoid content of the extract and subsequent fractions were determined spectrophotometrically. Ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis was used to tentatively identify the major phenolics in the most active fraction. RESULTS: The concentration of total phenolics and total flavonoids in the extract and each fraction thereof correlated with the level of carbonic anhydrase inhibition activity. The ethyl acetate fraction of the aqueous ethanol extract of L. acutangula had the highest carbonic anhydrase inhibition activity. The enzyme kinetics analysis indicated a mixed mode of inhibition. UPLC-QTOF-MS analysis of the ethyl acetate fraction indicated a number of phenolic acids, hydroxycoumarins, flavones, flavanones, and flavonoids. CONCLUSION: The correlation of total phenolic content with carbonic anhydrase inhibition suggested further research that might confirm that phenolic compounds of L. acutangula offer potential therapeutic benefits against carbonic anhydrase-related disorders.