Triggering Breast Cancer Apoptosis via Cyclin-Dependent Kinase Inhibition and DNA Damage by Novel Pyrimidinone and 1,2,4-Triazolo[4,3-a]pyrimidinone Derivatives.

Combinations of apoptotic inducers are common clinical practice in breast cancer. However, their efficacy is limited by the heterogeneous pharmacokinetic profiles. An advantageous alternative is merging their molecular entities in hybrid multitargeted scaffolds exhibiting synergistic activities and uniform distribution. Herein, we report apoptotic inducers simultaneously targeting DNA and CDK-2 (cyclin-dependent kinase-2) inspired by studies revealing that CDK-2 inhibition sensitizes breast cancer to DNA-damaging agents. Accordingly, rationally substituted pyrimidines and triazolopyrimidines were synthesized and assayed by MTT against MCF-7, MDA-MB231, and Wi-38 cells compared to doxorubicin. The N-(4-amino-2-((2-hydrazinyl-2-oxoethyl)thio)-6-oxo-1,6-dihydropyrimidin-5-yl)acetamide 5 and its p-nitrophenylhydrazone 8 were the study hits against MCF-7 (IC50 = 0.050 and 0.146 µM) and MDA-MB231 (IC50 = 0.826 and 0.583 µM), induced DNA damage at 10.64 and 30.03 nM, and inhibited CDK-2 (IC50 = 0.172 and 0.189 µM). 5 induced MCF-7 apoptosis by 46.75% and disrupted cell cycle during S phase. Docking and MD simulations postulated their stable key interactions.

Sensitive "release-on-demand" fluorescent genosensors for probing DNA damage induced by commonly used cardiovascular drugs: Comparative study.

Cardiovascular drugs (CVDs) are agents working on the heart and the vascular system to treat many cardiovascular disorders. Such disorders represent the leading cause for morbidity and mortality worldwide. The treatment regimen includes different administered drugs on chronic basis. The cumulative drugs in human body coincides with exposure to electromagnetic radiations from different sources leading to drug-radiation interaction that may lead to drug photosensitization. Such photosensitization may lead to mutagenesis, cancer, and cell death due to molecular damage to DNA. This work involves the application of two bioluminescent genosensors; Terbium chloride and EvaGreen are utilized to investigate potential DNA damage caused by frequently used CVDs following UVA irradiation. A variety of CVDs are investigated. Ten drugs; Amiloride, Atorvastatin, Captopril, Enalapril, Felodipine, Hydrochlorothiazide, Indapamide, Losartan, Triamterene and Valsartan are studied. The study's findings showed that such drugs induced DNA damage following UVA irradiation. The induced DNA damage altered the fluorescence of terbium chloride and EvaGreen genosensors, proportionally. The results are confirmed by viscosity measurements reflecting the possible intercalation of CVDs with DNA. Also, the work is applied on calf thymus DNA to mimic the actual biological variability. The demonstrated bioluminescent genosensors provide automatic, simple and low-cost methods for assessing DNA-drug interactions.

New imidazole-2-thiones linked to acenaphythylenone as dual DNA intercalators and topoisomerase II inhibitors: structural optimization, docking, and apoptosis studies.

In this article, a new series of 2-((3,5-disubstituted-2-thioxo-imidazol-1-yl)imino)acenaphthylen-1(2H)-ones were synthesized. Imidazole-2-thione with acenaphthylen-one gave a hybrid scaffold that integrated key structural elements essential for DNA damage via direct DNA intercalation and inhibition of the topoisomerase II enzyme. All the synthesized compounds were screened to detect their DNA damage using a terbium fluorescent probe. Results demonstrated that 4-phenyl-imidazoles 5b and 5e in addition to 4-(4-chlorophenyl)imidazoles 5h and 5j would induce detectable potent damage in ctDNA. The four most potent compounds as DNA intercalators were further evaluated for their antiproliferative activity against HepG2, MCF-7 and HCT-116 utilizing the MTT assay. The highest anticancer activity was recorded with compounds 5b and 5h against the breast cancer cell line MCF-7 which were 1.5- and 3- folds more active than doxorubicin, respectively. Therefore, imidazole-2-thione tethered acenaphthylenone derivatives can be considered as promising scaffold for the development of effective dual DNA intercalators and topoisomerase II inhibitors.

Identification of new 5-(2,6-dichlorophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-7-carboxylic acids as p38α MAPK inhibitors: Design, synthesis, antitumor evaluation, molecular docking and in silico studies.

In pursuit of discovering novel scaffolds that demonstrate potential inhibitory activity against p38α MAPK and possess strong antitumor effects, we herein report the design and synthesis of new series of 17 final target 5-(2,6-dichlorophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-7-carboxylic acids (4-20). Chemical characterization of the compounds was performed using FT-IR, NMR, elemental analyses and mass spectra of some representative examples. With many compounds showing potential inhibitory activity against p38α MAPK, two derivatives, 8 and 9, demonstrated the highest activity (>70 % inhibition) among the series. Derivative 9 displayed IC50 value nearly 2.5 folds more potent than 8. As anticipated, they both showed explicit interactions inside the kinase active site with the key binding amino acid residues. Screening both compounds for cytotoxic effects, they exhibited strong antitumor activities against lung (A549), breast (MCF-7 and MDA MB-231), colon (HCT-116) and liver (Hep-G2) cancers more potent than reference 5-FU. Their noticeable strong antitumor activity pointed out to the possibility of an augmented DNA binding mechanism of antitumor action besides their kinase inhibition. Both 8 and 9 exhibited strong ctDNA damaging effects in nanomolar range. Further mechanistic antitumor studies revealed ability of compounds 8 and 9 to arrest cell cycle in MCF-7 cells at S phase, while in HCT-116 treated cells at G0-G1 and G2/M phases. They also displayed apoptotic induction effects in both MCF-7 and HCT-116 with total cell deaths more than control untreated cells in reference to 5-FU. Finally, the compounds were tested for their anti-migratory potential utilizing wound healing assay. They induced a significant decrease in wound closure percentage after 24 h treatment in the examined cancer cells when compared to untreated control MCF-7 and HCT-116 cells better than 5-FU. In silico computation of physicochemical parameters revealed the drug-like properties of 8 and 9 with no violation to Lipinski's rule of five as well as their tolerable ADMET parameters, thus suggesting their utilization as potential future drug leads amenable for further optimization and development.

Discovery of novel benzimidazole acyclic C-nucleoside DNA intercalators halting breast cancer growth.

Breast cancer continues to be the most frequent cancer worldwide. In practice, successful clinical outcomes were achieved via targeting DNA. Along with the advances in introducing new DNA-targeting agents, the "sugar approach" design was employed herein to develop new intercalators bearing pharmacophoric motifs tethered to carbohydrate appendages. Accordingly, new benzimidazole acyclic C-nucleosides were rationally designed, synthesized and assayed via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay to evaluate their cytotoxicity against MCF-7 and MDA-MB-231 breast cancer cells compared to normal fibroblasts (Wi-38), compared to doxorubicin. (1S,2R,3S,4R)-2-(1,2,3,4,5-Pentahydroxy)pentyl-1H-5,6-dichlorobenzimidazole 7 and (1S,2R,3S,4R)-2-(1,2,3,4,5-pentahydroxy)pentyl-1H-naphthimidazole 13 were the most potent and selective derivatives against MCF-7 (half-maximal inhibitory concentration [IC50 ] = 0.060 and 0.080 µM, selectivity index [SI] = 9.68 and 8.27, respectively) and MDA-MB-231 cells (IC50 = 0.299 and 0.166 µM, SI = 1.94 and 3.98, respectively). Thus, they were identified as the study hits for mechanistic studies. Both derivatives induced DNA damage at 0.24 and 0.29 µM, respectively. The DNA damage kinetics were studied compared to doxorubicin, where they both induced faster damage than doxorubicin. This indicated that 7 and 13 showed a more potent DNA-damaging effect than doxorubicin. Docking simulations within the DNA double strands highlighted the role of both the heterocyclic core and the sugar side chain in exhibiting key H-bond interactions with DNA bases.

White sustainable luminescent determination of nifuroxazide using nitrogen-sulphur co-doped carbon quantum dots nanosensor in bulk and various pharmaceutical matrices.

Nifuroxazide (NFX) is an antimicrobial agent that is frequently used as an intestinal antiseptic and recently was proven to have anticancer properties. This work employs the use of nitrogen and sulphur co-doped carbon quantum dots (NSC-dots) luminescent nanoparticles to propose a highly sensitive, sustainable, white and green spectrofluorometric method for NFX detection in bulk and pharmaceutical dosage forms. l-Cysteine and citric acid were the precursors to synthesize water soluble NSC-dots by a quick and environmentally-friendly hydrothermal process. NSC-dots' native fluorescence was measured at λem = 416 nm following excitation at 345 nm. Addition of NFX resulted in quantitative quenching of NSC-dots' luminescence, which represents the principle over which this luminescent method was based. Additionally, the mechanism of fluorescence quenching was studied and discussed. The analytical procedure was validated according to the ICH-guidelines. Linear response for NFX was obtained in the dynamic range 0.04-15 µg mL-1. The estimated NFX detection and quantification limits were 0.005 and 0.015 µg mL-1, respectively. The proposed method was employed for NFX quantification into two commercial pharmaceutical dosage forms. The calculated percentage recoveries (R%), percentage relative standard deviations (RSD%), and percentage error (Er%) were satisfactory. Comparison with other reported methods showed that the proposed method is superior in several aspects. Evaluation of the whiteness of the proposed method using the RGB 12 algorithm combined with the most widely used greenness evaluation tools, the Analytical Eco-Scale and AGREE, demonstrated its superiority and sustainability over other previously published spectrofluorimetric methods for the assay of NFX in various dosage forms.

Multiple green spectroscopic methods for erdosteine determination in bulk and dosage form with extensive greenness evaluation.

Four simple, sensitive, economical, and eco-friendly spectrophotometric and spectrofluorimetric methods for the assay of erdosteine (ERD) in bulk and dosage form have been developed and validated as per the current ICH guidelines. Method I involved the addition of the powerful oxidizing agent, potassium permanganate to ERD and measuring the oxidation product at 600 nm. Another oxidizing agent; ceric ammonium sulfate was used in Method II where ERD is oxidized resulting in a decline in the absorbance intensity of cerium (IV) ions, measured at 320 nm. Similarly, Method III employed the use of ceric ammonium sulfate, However, the fluorescence intensity of the resulting cerium (III) ions was recorded at λex/λem 255/355 nm, respectively. Whereas in Method IV, ERD was added to acriflavine leading to a proportional decrease in its native fluorescence. Various reaction conditions affecting the intensity of measurement were attentively investigated, optimized, and validated. All the suggested methods did not require any tedious extraction procedures nor organic solvents. The implementation of the proposed methods in ERD assay resulted in linear relationships between the measured signals and the corresponding concentrations of ERD in the range of 1-6, 0.1-1.0, 0.01-0.1, and 10-100 µg/mL with LOD values 0.179, 0.024, 0.0027 and, 3.2 µg/mL for methods I, II, III and IV respectively. The suggested methods were successfully applied to ERD analysis in pure form and in commercial capsules. Furthermore, the eco-friendliness of the proposed methods was thoroughly checked using various greenness testing tools. Lastly, this work, not only presents highly sensitive, green, mix-and-read methods for ERD determination, but also, describes the determination of ERD spectrofluorimetrically for the first time in the literature.

Green "turn-off" luminescent nanosensors for the sensitive determination of desperately fluorescent antibacterial antiviral agent and its metabolite in various matrices.

Nitazoxanide (NTX) is an antimicrobial drug that was used for the treatment of various protozoa. However, during the coronavirus pandemic, NTX has been redirected for the treatment of such virus that primarily infect the respiratory tract system. NTX is now used as a broad-spectrum antiviral agent. In this study, a highly sensitive and green spectrofluorometric method was developed to detect NTX in various dosage forms and its metabolite, tizoxanide (TX), in human plasma samples using nitrogen and sulfur co-doped carbon quantum dots nanosensors (C-dots). A simple and eco-friendly hydrothermal method was used to synthetize water soluble C-dots from citric acid and l-cysteine. After excitation at 345 nm, the luminescence intensity was measured at 416 nm. Quenching of C-dots luminescence occurred upon the addition of NTX and was proportional to NTX concentration. Assessment of the quenching mechanism was performed to prove that inner filter effect is the underlying molecular mechanism of NTX quenching accomplished. After optimizing all experimental parameters, the analytical procedure was evaluated and validated using the ICH guidelines. The method linearity, detection and quantification limits of NTX were 15 × 10-3-15.00 µg/mL, 56.00 × 10-4 and 15 × 10-3 µg/mL, respectively. The proposed method was applied for the determination of NTX in its commercial pharmaceutical products; Nanazoxid® oral suspension and tablets. The obtained % recovery, relative standard deviation and % relative error were satisfactory. Comparison with other reported spectrofluorimetric methods revealed the superior sensitivity of the proposed method. Such high sensitivity permitted the selective determination of TX, the main metabolite of NTX, in human plasma samples making this study the first spectrofluorimetric method in literature that determine TX in human plasma samples. Moreover, the method greenness was assessed using both Eco-Scale and AGREE approaches to prove the superiority of the proposed method greenness over other previously published spectrofluorimetric methods for the analysis of NTX and its metabolite, TX, in various dosage forms and in human plasma samples.

Nicorandil/ morphine crosstalk accounts for antinociception and hepatoprotection in hepatic fibrosis in rats: Distinct roles of opioid/cGMP and NO/KATP pathways.

Previous report indicated that nicorandil potentiated morphine antinociception and attenuated hepatic injury in liver fibrotic rats. Herein, the underlying mechanisms of nicorandil/morphine interaction were investigated using pharmacological, biochemical, histopathological, and molecular docking studies. Male Wistar rats were injected intraperitoneally (i.p.) with carbon tetrachloride (CCl4, 40%, 2 ml/kg) twice weekly for 5 weeks to induce hepatic fibrosis. Nicorandil (15 mg/kg/day) was administered per os (p.o.) for 14 days in presence of the blockers; glibenclamide (KATP channel blocker, 5 mg/kg, p.o.), L-NG-nitro-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor, 15 mg/kg, p.o.), methylene blue (MB, guanylyl cyclase inhibitor, 2 mg/kg, i.p.) and naltrexone (opioid antagonist, 20 mg/kg, i.p.). At the end of the 5th week, analgesia was evaluated using tail flick and formalin tests along with biochemical determinations of liver function tests, oxidative stress markers and histopathological examination of liver tissues. Naltrexone and MB inhibited the antinociceptive activity of the combination. Furthermore, combined nicorandil/morphine regimen attenuated the release of endogenous peptides. Docking studies revealed a possible interaction of nicorandil on µ, κ and δ opioid receptors. Nicorandil/morphine combination protected against liver damage as evident by decreased liver enzymes, liver index, hyaluronic acid, lipid peroxidation, fibrotic insults, and increased superoxide dismutase activity. Nicorandil/morphine hepatoprotection and antioxidant activity were inhibited by glibenclamide and L-NAME but not by naltrexone or MB. These findings implicate opioid activation/cGMP versus NO/KATP channels in the augmented antinociception, and hepatoprotection, respectively, of the combined therapy and implicate provoked cross talk by nicorandil and morphine on opioid receptors and cGMP signaling pathway. That said, nicorandil/morphine combination provides a potential multitargeted therapy to alleviate pain and preserve liver function.

Recent analytical methodologies for the determination of anti-covid-19 drug therapies in various matrices: a critical review.

Since the discovery of the first case infected with severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) in Wuhan, China in December 2019, it has turned into a global pandemic. According to the World Health Organization (WHO) statistics, about 603.7 million confirmed coronavirus cases and 6.4 million deaths have been reported. Remdesivir (RMD) was the first U.S. Food and Drug Administration (FDA) approved antiviral drug for the treatment of coronavirus in pediatrics and adults with different disease severities, ranging from mild to severe, in both hospitalized and non-hospitalized patients. Various drug regimens are used in Covid-19 treatment, all of which rely on the use of antiviral agents including ritonavir (RTN)/nirmatrelvir (NTV) combination, molnupiravir (MLP) and favipiravir (FVP). Optimizing analytical methods for the selective and sensitive quantification of the above-mentioned drugs in pharmaceutical dosage forms and biological matrices is a must in the current pandemic. Several analytical techniques were reported for estimation of antivirals used in Covid-19 therapy. Chromatographic methods include Thin Layer Chromatography (TLC) densitometry, High Performance Thin Layer Chromatography (HPTLC), Reversed Phase-High Performance Liquid Chromatography (RP-HPLC), High Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC-MS/MS) or Ultraviolet detectors (HPLC-UV), Ultra High-Performance Liquid Chromatography (UHPLC-MS/MS) or (UPLC-UV) and Micellar Liquid Chromatography (MLC). In addition to other spectroscopic methods including Paper Spray Mass Spectrometry (PS-MS), UV-Visible Spectrophotometry, and Spectrofluorimetry. Herein, we will focus on the clarification of trendy, simple, rapid, accurate, precise, sensitive, selective, and eco-friendly analytical methods used for the analysis of anti-Covid-19 drugs in dosage forms as well as biological matrices.

Sensitive MALDI-TOF MS and 'turn-on' fluorescent genosensor for the determination of DNA damage induced by CNS acting drugs.

The genotoxic and carcinogenic adverse effects of various drugs should be considered for assessing drug benefit/risk ratio. On that account, the scope of this study is to examine the kinetics of DNA damage triggered by three CNS acting drugs; carbamazepine, quetiapine and desvenlafaxine. Two precise, simple and green approaches were proposed for probing drug induced DNA impairment; MALDI-TOF MS and terbium (Tb3+) fluorescent genosensor. The results revealed that all the studied drugs induced DNA damage manifested by the MALDI-TOF MS analysis as a significant disappearance of the DNA molecular ion peak with the appearance of other peaks at smaller m/z indicating the formation of DNA strand breaks. Moreover, significant enhancement of Tb3+ fluorescence occurred, proportional to the amount of DNA damage, upon incubation of each drug with dsDNA. Furthermore, the DNA damage mechanism is examined. The proposed Tb3+ fluorescent genosensor showed superior selectivity and sensitivity and is significantly simpler and less expensive than other methods reported for the detection of DNA damage. Moreover, the DNA damaging potency of these drugs was studied using calf thymus DNA in order to clarify the potential safety hazards associated with the studied drugs on natural DNA.

Inclusion of Nitrofurantoin into the Realm of Cancer Chemotherapy via Biology-Oriented Synthesis and Drug Repurposing.

Structural modifications of the antibacterial drug nitrofurantoin were envisioned, employing drug repurposing and biology-oriented drug synthesis, to serve as possible anticancer agents. Eleven compounds showed superior safety in non-cancerous human cells. Their antitumor efficacy was assessed on colorectal, breast, cervical, and liver cancer cells. Three compounds induced oxidative DNA damage in cancer cells with subsequent cellular apoptosis. They also upregulated the expression of Bax while downregulated that of Bcl-2 along with activating caspase 3/7. The DNA damage induced by these compounds, demonstrated by pATM nuclear shuttling, was comparable in both MCF7 and MDA-MB-231 (p53 mutant) cell lines. Mechanistic studies confirmed the dependence of these compounds on p53-mediated pathways as they suppressed the p53-MDM2 interaction. Indeed, exposure of radiosensitive prostatic cancer cells to low non-cytotoxic concentrations of compound 1 enhanced the cytotoxic response to radiation indicating a possible synergistic effect. In vivo antitumor activity was verified in an MCF7-xenograft animal model.

Novel genosensor for probing DNA mismatches and UV-induced DNA damage: Sequence-specific recognition.

Human genome is continuously susceptible to changes that may lead to undesirable mutations causing various diseases and cancer. Vast majority of techniques has investigated the discrimination between base-pair mismatched nucleic acid, but many of these techniques are time-consuming, complex, expensive, and limited to the detection of specific type of dsDNA mismatches. In this study, we introduce a simple mix-and-read assay for the sensitive and cost-effective analysis of DNA base mismatches and UV-induced DNA damage using Hoechst genosensor dye (H258). This dye is a minor groove binder that undergoes a drastic conformational change upon binding with mismatch DNA. The difference in binding affinity between perfectly matched and mismatched DNA was studied for sequences at different base mismatch locations and finally, extended for the detection of dsDNA damage by UVC radiation in calf thymus DNA. In addition, a comparative DNA damage kinetic study was performed using H258 (minor groove binder) and EvaGreen (intercalating) dye to get insight on assay selectivity and sensitivity with dye binding mechanism. The result shows good reproducibility making H258 genosensor a cheaper alternative for DNA mismatch and damage studies with possibility of extension for in-vitro detection of hot spots of DNA mutations.

Two novel "release-on-demand" fluorescent biosensors for probing UV-induced DNA damage induced in single stranded and double stranded DNA: Comparative study.

Light in the UVC spectral region damages both single-strand (ssDNA) and double-strand DNA (dsDNA), and contributes to the formation of mutagenic photoproducts. In-vivo studies show greater damage for ssDNA compared to dsDNA. However, excited-state spectroscopy shows that dsDNA has longer excited-state lifetime than ssDNA, which increases the probability of damage for dsDNA. However, lack of a direct comparison of in-vitro ssDNA and dsDNA damage rates precludes the development of a model that elucidates the molecular factors responsible for damage. In this work, two novel sensitive "release-on-demand" biosensors are developed for the selective probing of DNA-damage and comparing the rate of DNA damage in ssDNA and dsDNA. The two biosensors involve the use of EvaGreen and Hoechst dyes for the sensitive probing of DNA-damage. The results show that ssDNA is damaged at a faster rate than dsDNA in the presence of UVC light (200-295 nm). Furthermore, we examined the effect of G/C composition on the damage rate for mostly A/T ssDNA and dsDNA oligonucleotides. Our results show that DNA damage rates are highly dependent on the fraction of guanines in the sequence, but that in-vitro dsDNA always exhibits an overall slower rate of damage compared to ssDNA, essentially independent of sequence.

Simple simultaneous determination of moxifloxacin and metronidazole in complex biological matrices.

A simple, sensitive and rapid RP-HPLC method is presented, for the first time, for the simultaneous determination of moxifloxacin hydrochloride and metronidazole in different biological fluids including saliva and plasma without any matrix interference. The separation was performed using ACN and phosphate buffer (30 : 70% v/v) as the mobile phase on a Zorbax Eclipse Plus-C18 column attached to a guard column. The method was validated according to the FDA guidelines for bioanalytical method validation and was successfully applied for simultaneous determination of the studied drugs in saliva and plasma samples. The good precision and selectivity of the developed method allow it to be used for routine therapeutic drug monitoring of such drugs and it presents a simple and sensitive analytical tool for performing versatile pharmacokinetics and bioavailability studies. A DAD detector is valuable to determine each drug at its maximum wavelength to ensure high sensitivity. Determination of such a combination in saliva introduces a quick and non-invasive alternative to blood analysis.

Inexpensive bioluminescent genosensor for sensitive determination of DNA damage induced by some commonly used sunscreens.

Sunscreens (SSs) are highly applied all over the world on large areas of human body. Benzophenone chemical group constitute a major part in most SSs. Benzophenones are reported to induce changes in nucleic acids upon UV-irradiation. These alterations may potentially lead to DNA mutation, cell apoptosis, and eventually skin cancer. This work compares the kinetics of the induced DNA damage by some SSs after UV-irradiation. Six commonly used SSs; 4-t-butyl-4-methoxy dibenzoyl methane, 4-methoxycinnamic acid, 2-hydroxy-4-methoxybenzophenone (BZ-3), Dibenzoyl methane, 2,2'-dihydroxy-4-methoxy benzophenone (BZ-8) and p-methylbenzoic acid; are investigated. In this work, terbium chloride bioluminescent genosensor is used for sensitive, simple and inexpensive determination of induced DNA-damage. Results reveal that only BZ-3 and BZ-8 induced DNA-damage upon UV-irradiation that are confirmed by both absorption spectroscopy and viscosity measurements. Moreover, viscosity studies indicated the possible intercalation of the SS into DNA prior to initiation of DNA damage. Furthermore, the potency of BZ-3 and BZ-8 to induce DNA damage upon UVA irradiation was assessed on calf thymus DNA. The low cost of the proposed bioluminescent genosensor allows it to be an automatic simple process for the investigation of any DNA-drug interactions without the need of coupling with other analytical methods.

Simultaneous Quantitation of Paracetamol and Lornoxicam in the Presence of Five Related Substances and Toxic Impurities by a Selective HPLC-DAD Method.

OBJECTIVE: This research describes the simultaneous quantitation of paracetamol (PRM) and lornoxicam (LRX) with five of their related substances and toxic impurities, including, 4-nitrophenol (NTP), 4-aminophenol (AMP), 4-chloroacetanilide (CAC), N-phenylacetamide (NPA), and 2-aminopyridine (APD) using a specific HPLC-diode array detector (DAD) method. METHODS: The chromatographic separation involves the use of a XTerra C18 column as the stationary phase and a mobile phase consisting of acetonitrile and 0.025 M phosphate buffer (pH 6). The separation was performed using gradient elution mode at 1.0 mL/min flow rate and detection at 260 nm for the determination of PRM and LRX. For detecting PRM and LRX in the presence of their toxic impurities, 270 nm was used. Validation of the suggested HPLC method was accomplished with regard to linearity, ranges, detection and quantitation limits, robustness, accuracy, precision, and specificity. RESULTS: Excellent resolution of the mixture components was accomplished at retention times 4.2, 4.8, 7.4, 11.1, 13.5, 14.7, and 15.3 min for APD, AMP, PRM, NPA, LRX, NTP, and CAC, respectively. Linearity was established for PRM and LRX within concentration ranges of 10-100 and 10-60 µg/mL, respectively. The correlation coefficients obtained were >0.9997. The suggested method was confirmed to be a specific stability-indicating through the selective separation of PRM and LRX from their related substances, degradants, and impurities. CONCLUSION: The proposed method was successfully utilized for the sensitive and selective determination of PRM and LRX in their pharmaceutical formulation. HIGHLIGHTS: To the best of our knowledge, this is the first impurity profiling assay method for this combination in the presence of five of their toxic related substances and impurities. Taking into consideration that at least two of the studied impurities (AMP and APD) are actually reported degradation products for the main drugs, the suggested method can be considered stability-indicating as well.

Investigation of nucleic acid damage induced by a novel ruthenium anti-cancer drug using multiple analytical techniques: Sequence specificity and damage kinetics.

Cis-diacetonitrilo-bis(bipyridine) ruthenium(II) chloride is a recently introduced cis-platin analogue that has anti-cancer properties with lower side effects. However, the sequence dependence of its DNA damaging mechanism is unclear. Here, we present a simple, sensitive, multiplexed mix-and-read assay for ascertaining the molecular mechanism of DNA damage induced by the studied ruthenium complex (Ru-complex). The damage kinetics and sequence specificity for the Ru-complex induced DNA damage are examined by studying the induced damage in various oligonucleotide sequences by EvaGreen-DNA intercalator probe. High-through-put measurements were established using a 96-well microplate platform that allows multiple sequences to be measured simultaneously. The results show that the extent of damage increases with an increasing number of guanines, with considerable amount of damage at GA, GT and GC sites, in particular. Furthermore, the interaction of Ru-complex with DNA was confirmed using thermal analysis and MALDI-TOF-MS. Results indicate that the activated Ru-complex preferentially binds via both mono- and di-adduct formation at G and GG sites, respectively. Moreover, the developed method was successfully applied for the determination of the potency of the studied Ru-complex to induce DNA damage in K-Ras and N-Ras family of genes, one of the most common oncogenic events in cancer.

New chalcone-tethered 1,3,5-triazines potentiate the anticancer effect of cisplatin against human lung adenocarcinoma A549 cells by enhancing DNA damage and cell apoptosis.

In the pursuit of new compounds for co-treatment to enhance the anticancer efficacy of cisplatin against lung adenocarcinoma, a series of chalcone-tethered 1,3,5-triazines was designed and synthesized. MTT assay was used to evaluate the anticancer activity of the combinations in which two hybrids 10 and 12 were found to significantly inhibit A549 cancer cells viability and their IC50 values were 24.5 and 17 µM, respectively in reference to cisplatin (IC50 = 21.5 µM). The combined effect of cisplatin with each of 10 and 12 was analyzed according to Chou-Talalay method against both A549 and normal human fibroblast cells. Mechanistic studies employing MALDI-TOF MS and fluorescence spectroscopy using Evagreen probe inferred that 10 and 12 induced DNA double strand breaks in contrast to cisplatin which induces DNA interstrand cross-links. Also, DNA damage kinetics study demonstrated the difference in the rate of DNA damage induced by both 10 and 12 alone and in combination with cisplatin. Further Annexin V-FITC/propidium iodide dual staining assay provided evidence that 10 and 12 induced apoptosis via different pattern to cisplatin and their combination with cisplatin promoted more cells to enter late apoptosis and necrosis. Molecular docking of 10 and 12 in the active pocket of DNA dodecamer displayed their binding modes with higher number of stable hydrogen bond donor as well as π-H interactions in reference to the original ligand.

Green analytical methods for simultaneous determination of compounds having relatively disparate absorbance; application to antibiotic formulation of azithromycin and levofloxacin.

Green validated spectrophotometric methods are developed for simultaneous determination of Azithromycin (AZI) and Levofloxacin (LEVO) antibiotic mixture. Determination of AZI presents a real analytical challenge as its structure lacks any chromophore, and hence it cannot be determined by direct spectrophotometry. However, the reaction of AZI with perchloric acid produces a green product that can be accurately determined spectrophotometrically. Thus, the work presented demonstrates simple green and sensitive methods for the simultaneous determination of AZI and LEVO mixture. Method I depends on direct measurement of absorbance of azithromycin and levofloxacin in perchloric acid methanolic solution at 482 nm and 224 nm, respectively. While, Method II depends on measuring the first derivative spectrophotometric peak-to-peak amplitudes of AZI and LEVO in perchloric acid methanolic solution at 475-490 nm and 280-253 nm, respectively. Regression analysis shows good linearity for AZI and LEVO over the concentration ranges of 5-50 and 2.5-20 µg/mL for method I and 5-50 and 5-40 µg/mL for method II for AZI and LEVO, respectively. The proposed methods were validated in compliance with ICH guidelines. The suggested procedures are successfully applied for the assay of AZI and LEVO mixture in bulk powder and laboratory-prepared tablets. Greenness profile of the proposed methods were compared with other published methods through applying the Eco-scale protocol. Assessment results demonstrated that the proposed methods are greener than other reported methods. Moreover, upon comparison with other methods, the proposed methods showed better or comparable sensitivity in addition to being selective and rapid with no requirement for laborious extraction techniques. These advantages encourage the application of the proposed methods in routine analysis of AZI and LEVO in quality control laboratories as green and simple analytical tool.