Identification, Isolation, and Characterization of a Novel Degradation Impurity of Busulfan Using Preparative Chromatography, NMR, and LC-MS.

BACKGROUND: Busulfan is the most effective medication for treating chronic myelogenous or granulocytic leukemia because it has cytotoxic properties that harm or kill hematopoietic cells. It cannot absorb light in the Ultraviolet range due to its structure. Because of this, it is very challenging to quantify using traditional HPLC coupled with UV/Photodiode Array detectors. So, using sodium diethyldithiocarbamate, a derivatization method was developed to quantify related impurities. A significant unknown impurity was identified in derivatized samples of busulfan and a noticeably high percentage level was discovered during routine drug testing. OBJECTIVE: We aimed to isolate, and characterize the unknown impurity observed in the samples and to identify its root cause. METHODS: Preparative HPLC was used to isolate the unidentified, derivatized impurity, and 1H NMR, 13C NMR, and MS were used to decipher its structural components. RESULTS: The spectral characterization data analysis showed that the unknown impurity was related to busulfan. Additionally, it was noted that the impurity developed as a result of the residual buffer used to prepare the derivatizing reagent. CONCLUSION: The isolated impurity was found to be same as comparable to that found in busulfan drug substances, according to the results of the characterization tools. An alternative method of reagent preparation was optimized and deemed satisfactory because the buffer used in reagent preparation is the only factor contributing to the formation of impurities. HIGHLIGHTS: Using cutting-edge analytical characterization tools, it was possible to explain the structural characteristics of an unknown impurity and discover that it was a novel impurity, which undoubtedly contributes to the comprehension of drug substance reaction properties.

Stability-Indicating Method Development and Validation for Busulfan Drug Substance by Gas Chromatography-Flame Ionization Detector.

A new, simple and stability-indicating gas chromatography-flame ionization detection (GC-FID) method was developed and validated for the quantitative determination of busulfan and its organic impurities (OI) in drug substance without derivatization. The chromatographic attributes were achieved on a fused silica capillary column (0.53 mm × 30 m, 1.0 µm, USP Phase G42), using hydrogen as a carrier gas with a split ratio of 1:1. Forced degradation studies were conducted to establish the stability-indicating capability and method specificity that showed the stressed busulfan peak was free from any co-elution. Robustness study demonstrated the chromatograms remained mostly unaffected under deliberate, but small variations of chromatographic parameters, establishing the reliability of the method during routine usage. The method was shown to be reliable, sensitive, specific, linear, accurate, precise and rugged in the 1,4-butanediol concentration range of 1-20 µg/mL. The method, intended for compendial uses, is suitable for quantitative analysis of busulfan and its organic impurities in drug substances.

Electrophilic reactivity of the Busulfan metabolite, EdAG, towards cellular thiols and inhibition of human thioredoxin-1.

Busulfan is an alkylating agent used in chemotherapy conditioning regimens prior to hematopoietic stem cell transplantation (HSCT). However, its administration is associated with a great risk of adverse toxicities, which have been historically attributed to busulfan's mechanism of non-specific DNA alkylation. A phase II generated metabolite of busulfan, EdAG (γ-glutamyldehydroalanylglycine), is a dehydroalanine analog of glutathione (GSH) with an electrophilic moiety, suggesting it may bind to proteins and disrupt biological function. However, EdAG's reactions with common cellular thiols such as glutathione (GSH) and l-cysteine are understudied, along with possible inhibition of glutathionylation-dependent enzymes (with active site cysteine residues). We established a physiologically-relevant in vitro model to readily measure thiol loss over time. Using this model, we compared the apparent rates of thiol depletion in the presence of EdAG or arecoline, a toxic constituent of the areca (betel) nut and known GSH depletor. Simulated kinetic modeling revealed that the mean (±SE) alpha (α) second order rate constants describing GSH and l-cysteine depletion in the presence of EdAG were 0.00522 (0.00845) µM-1∙min-1 and 0.0207 (0.00721) µM-1∙min-1, respectively; in the presence of arecoline, the apparent rates of depletion were 0.0619 (0.009) µM-1∙min-1 and 0.2834 (0.0637) µM-1∙min-1 for GSH and l-cysteine, respectively. Under these experimental conditions, we conclude that EdAG was a weaker electrophile than arecoline. Arecoline and EdAG both depleted apparent l-cysteine concentrations to a much greater extent than GSH, approximately 4.58-fold and 3.97-fold change greater, respectively. EdAG modestly inhibited (∼20%) the human thioredoxin-1 (hTrx-1) catalyzed reduction of insulin with a mean IC50 of 93 µM [95% CI: 78.6-110 µM). In summary, EdAG's ability to spontaneously react with endogenous thiols and inhibit hTrx-1 are potentially biochemically relevant in humans. These findings continue to support the growing concept that EdAG, an underrecognized phase II metabolite of busulfan, plays a role in untoward cellular toxicities during busulfan pharmacotherapy.

Docking analysis of aryl derivatives of diepoxide alkylating agents.

In this research, molecular structural manipulation of treosulfan alkylating agent and resultant changes in binding is studied to assist in designing derivatives of treosulfan for synthesis. Molecular docking has been conducted on simulated heterocyclic polyaromatic alkylating diepoxide derivatives of treosulfan with DNA nucleobases of dodecamer duplex of sequences d(CGCGAATTCGCG) and d(CGCGAATTCGCG) using Autodock vina package. Two series of simulated diepoxide molecules were designed with increasing aryl ring chain in linear and fused aryl way between the two epoxide reactive rings. Relationship between increasing no. of aryl rings (both linear and fused) between epoxide moieties on the binding energy values was evaluated. We also identified that designed molecules bind specifically to Guanine and Cytosine (GC) base pairs on DNA. Mode of interaction and resultant behavior as an alkylating agent or as minor groove binder was also found to be dependent up on the no. of aryl rings and their connectivity in the molecule. Both linearly bonded and fused aryl rings in higher number, between the epoxide rings, gave the strongest binding with the binding energy up to -8.1 and -8.7 Kcal/mol, respectively. These relationships can immensely help in designing and synthesis of derivatives of treosulfan like diepoxide based alkylating agents.

Endocrine and local signaling interact to regulate spermatogenesis in zebrafish: follicle-stimulating hormone, retinoic acid and androgens.

Retinoic acid (RA) is crucial for mammalian spermatogonia differentiation, and stimulates Stra8 expression, a gene required for meiosis. Certain fish species, including zebrafish, have lost the stra8 gene. While RA still seems important for spermatogenesis in fish, it is not known which stage(s) respond to RA or whether its effects are integrated into the endocrine regulation of spermatogenesis. In zebrafish, RA promoted spermatogonia differentiation, supported androgen-stimulated meiosis, and reduced spermatocyte and spermatid apoptosis. Follicle-stimulating hormone (Fsh) stimulated RA production. Expressing a dominant-negative RA receptor variant in germ cells clearly disturbed spermatogenesis but meiosis and spermiogenesis still took place, although sperm quality was low in 6-month-old adults. This condition also activated Leydig cells. Three months later, spermatogenesis apparently had recovered, but doubling of testis weight demonstrated hypertrophy, apoptosis/DNA damage among spermatids was high and sperm quality remained low. We conclude that RA signaling is important for zebrafish spermatogenesis but is not of crucial relevance. As Fsh stimulates androgen and RA production, germ cell-mediated, RA-dependent reduction of Leydig cell activity may form a hitherto unknown intratesticular negative-feedback loop.

Novel Pyrene Excimer and Fluorogenic Probe for the Detection of Alkylating Agents.

A pyrene-containing salicylic acid derivative (4) was found to be low in fluorescence, but its derivative pyrene-containing methyl salicylate (3) was found to be highly fluorescent in aqueous solution. This derivative has been tested in solution and found to be superior in the fluorogenic assay of pharmaceutical compounds, detection of chemical warfare agents, a preliminary toxicology test, mutagenicity of medicinal compounds, and other chemical analyses, including trimethylsilyl diazomethane; alkyl bromides and iodides; a sulfur mustard mimic 2-chloroethyl ethyl sulfide; and anticancer drugs, busulfan and pipobroman. The salicylic acid derivative (4) was applied as a fluorogenic probe for the detection of alkylating agents by esterification and generating fluorescence at 475 nm in solutions at low concentrations.

Kinetics of in Vitro Guanine- N7-Alkylation in Calf Thymus DNA by (2 S,3 S)-1,2-Epoxybutane-3,4-diol 4-methanesulfonate and (2 S,3 S)-1,2:3,4-Diepoxybutane: Revision of the Mechanism of DNA Cross-Linking by the Prodrug Treosulfan.

Prodrug treosulfan, originally registered for treatment of ovarian cancer, has gained a use in conditioning prior to hematopoietic stem cell transplantation. Treosulfan converts nonenzymatically to the monoepoxide intermediate (EBDM), and then to (2 S,3 S)-1,2:3,4-diepoxybutane (DEB). The latter alkylates DNA forming mainly (2' S,3' S)- N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (THBG) and (2 S,3 S)-1,4-bis(guan-7'-yl)butane-2,3-diol cross-link (bis-N7G-BD) via the intermediate epoxide adduct (EHBG). It is believed that DNA cross-linking by DEB is a primary mechanism for the anticancer and myeloablative properties of treosulfan, but clear evidence is lacking. Recently, we have proved that EBDM alkylates DNA producing (2' S,3' S)- N-7-(2',3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)-guanine (HMSBG) and that free HMSBG converts to EHBG. In this paper, we investigated the kinetics of HMSBG, bis-N7G-BD, and THBG in DNA in vitro to elucidate the contribution of EBDM and DEB to treosulfan-dependent DNA-DNA cross-linking. Calf thymus DNA was exposed to ( A) 100 µM treosulfan, ( B) 200 µM treosulfan, and ( C) DEB at a concentration 100 µM, exceeding that produced by 200 µM treosulfan. Following mild acid thermal hydrolysis of DNA, ultrafiltration, and off-line HPLC purification, the guanine adducts were quantified by LC-MS/MS. Both bis-N7G-BD and THBG reached highest concentrations in the DNA in experiment B. Ratios of the maximal concentration of bis-N7G-BD and THBG to DEB (adduct Cmax/DEB Cmax) in experiments A and B were 1.7-3.0-times greater than in experiment C. EHBG converted to the bis-N7G-BD cross-link at a much higher rate constant (0.20 h-1) than EBDM and DEB initially alkylated the DNA (1.8-3.4 × 10-5 h-1), giving rise to HMSBG and EHBG, respectively. HMSBG decayed unexpectedly slowly (0.022 h-1) compared with the previously reported behavior of the free adduct (0.14 h-1), which revealed the inhibitory effect of the DNA environment on the adduct epoxidation to EHBG. A kinetic simulation based on the obtained results and the literature pharmacokinetic parameters of treosulfan, EBDM, and DEB suggested that in patients treated with the prodrug, EBDM could produce the vast majority of EHBG and bis-N7G-BD via HMSBG. In conclusion, EBDM can produce DNA-DNA lesions independently of DEB, and likely plays a greater role in DNA cross-linking after in vivo administration of treosulfan than DEB. These findings compel revision of the previously proposed mechanism of the pharmacological action of treosulfan and contribute to better understanding of the importance of EBDM for biological effects.

Liquid chromatography-tandem mass spectrometry method for simultaneous determination of three N-7-guanine adducts of the active epoxides of prodrug treosulfan in DNA in vitro.

Prodrug treosulfan undergoes a pH and temperature-dependent activation to the monoepoxide intermediate (EBDM) and (2S,3S)-1,2:3,4-diepoxybutane (DEB). The latter DNA cross-linker is presently believed to mainly account for the pharmacological action of treosulfan. However, neither respective monoadducts nor cross-links have been isolated from treosulfan-treated DNA, and the exact alkylation mechanism of the treosulfan epoxides is unclear. In this paper, liquid chromatography method with tandem mass spectrometry detection (LC-MS/MS) for simultaneous determination of the N-7-guanine adducts of EBDM and DEB - (2'S,3'S)-N-7-(2'3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)guanine (HMSBG), N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (THBG), and 1,4-bis(N-7-guanyl)butane-2,3-diol cross-link (bis-N7G-BD) - in calf-thymus DNA has been developed and validated for the first time. The mixture of drug-free nucleic acid with the analytes and 15N-isotope labeled internal standards underwent a mild acid thermal hydrolysis and ultrafiltration (cut-off 10 kDa). Following offline LC purification, the analytes and internal standards were determined in the LC-MS/MS system with an electrospray interface. Complete resolution of THBG, HMSBG, and bis-N7G-BD was accomplished on a Zorbax Eclipse C18 column using gradient elution with a mobile phase composed of 0.1% formic acid and acetonitrile. Calibration curves were linear in the ranges: THBG 0.2-200 pmol, HMSBG 0.2-20 pmol, and bis-N7G-BD 0.4-40 pmol. The limits of quantitation allowed to determine the adducts at concentration of 330 or 660 per 109 DNA nucleotides. The LC-MS/MS method was adequately precise (coefficient of variation ≤ 16.7%) and accurate (relative error ≤ 17.7%). Calibration standards were stable for 14 days at -25 °C. The validated method enabled determination of THBG, HMSBG, and bis-N7G-BD in calf thymus DNA treated with treosulfan at pH 7.2 and 37 °C, which constitutes a novel bioanalytical application. To the authors' best knowledge, the quantification of THBG and bis-N7G-BD in one analytical run is also reported for the first time.

Pre-formulation investigations for establishing a protocol for treosulfan handling and activation.

INTRODUCTION: Treosulfan is an alkylating agent that is used for the treatment of ovarian cancer and for conditioning prior to stem cell transplantation. It is a prodrug that is activated non-enzymatically to two active epoxides. OBJECTIVES: To optimize a protocol for both in vivo samples handling and in vitro drug preparation. Treosulfan stability was tested in biological fluids at different conditions as well as for its cytotoxicity on cell lines. RESULTS: Plasma samples can be safely frozen for a short period up to 8 h, however; for longer periods, samples should be acidified. Urine samples and cell culture media can be safely frozen regardless their pH. For in vitro investigations, incubation of treosulfan at 37 °C for 24 h activated 100% of the drug. Whole blood acidification should be avoided for the risk of hemolysis. Finally; treosulfan cytotoxicity on HL-60 cells has increased following pre-incubation for 24 h at 37 °C compared to K562 cell line. CONCLUSION: The stability profiling of treosulfan provided a valuable reference for handling of biological samples for both in vivo and in vitro studies. These results can be utilized for further investigations concerning the drug kinetics and dynamics in addition to the development of new pharmaceutical formulations.

N-7-Guanine Adduct of the Active Monoepoxide of Prodrug Treosulfan: First Synthesis, Characterization, and Decomposition Profile Under Physiological Conditions.

(2S,3S)-1,2:3,4-diepoxybutane (DEB) cross-links DNA guanines by forming the intermediate epoxy-adduct ((2'S,3'S)-N-7-(3',4'-epoxy-2'-hydroxybut-1'-yl)guanine [EHBG]). This process is presently considered a primary mechanism for the action of treosulfan (TREO), the prodrug that transforms to DEB via the monoepoxide intermediate (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate (EBDM). In this article, the N-7-guanine adduct of EBDM ((2'S,3'S)-N-7-(2'3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)guanine [HMSBG]) was synthesized for the first time, and its stability was investigated at physiological in vitro conditions. To synthesize HMSBG, EBDM, formed in-situ from TREO, was treated with guanosine in glacial acetic acid at 60°C followed by ribose cleavage in 1 M HCl at 80°C. HMSBG was stable during the synthesis, which showed that a ß-hydroxy group protects the sulfonate moiety against hydrolysis in acid environment. At pH 7.2 and 37°C, HMSBG exclusively underwent first-order epoxidation to EHBG with a half-life of 5.0 h. EHBG further decomposed to trihydroxybutyl-guanine, chlorodihydroxybutyl-guanine (major products), phosphodihydroxy-guanine, and a structural isomer (minor products). The isomeric derivative was identified as guanine with a fused 7-membered ring, which provided a new insight into the EHBG stability. To conclude, the exclusive conversion of HMSBG to EHBG indicates that EBDM might contribute to DNA cross-linking independently from DEB and play a more important role in the TREO action than expected before.

Aryl sulfonate based anticancer alkylating agents.

This research work revolves around synthesis of antineoplastic alkylating sulfonate esters with dual alkylating sites for crosslinking of the DNA strands. These molecules were evaluated as potential antineoplastic cross linking alkylating agents by reaction with the nucleoside of Guanine DNA nucleobase at both ends of the synthesized molecule. Synthesis of the alkylating molecules and the crosslinking with the guanosine nucleoside was monitored by MALDITOF mass spectroscopy. The synthesized molecule's crosslinking or adduct forming rate with the nucleoside was compared with that of 1,4 butane disulfonate (busulfan), in form of time taken for the appearance of [M+H]+. It was found that aryl sulfonate leaving group was causing higher rate of nucleophilic attack by the Lewis basic site of the nucleobase. Furthermore, the rate was also found to be a function of electron withdrawing or donating nature of the substituent on the aryl ring. Compound with strong electron withdrawing substituent on the para position of the ring reacted fastest. Hence, new alkylating agents were synthesized with optimized or desired reactivity.

Development and characterization of an organic solvent free, proliposomal formulation of Busulfan using quality by design approach.

Parenteral administration of Busulfan (BU) conquers the bioavailability and biovariability related issues of oral BU by maintaining the plasma drug concentration in therapeutic range with minimal fluctuations thereby significantly reducing the side effects. Busulfex® is the only commercially available parenteral formulation of BU composed of organic solvents N, N-dimethylacetamide and polyethylene glycol 400. Since, BU is highly susceptible to hydrolytic degradation; Busulfex® has poor physical and chemical stability in IV fluids. It is quintessential to develop organic solvent free formulation of BU using parenterally acceptable excipients to enhance its solubility and stability in IV fluids. The Proliposomal formulation of BU was prepared by adsorption-sonicaton method using egg phosphotidylcholine, cholesterol, tween 80 and mannitol. Vesicle size and entrapment efficiency were optimized using 24 full factorial design and characterized by DSC, PXRD and TEM. Optimized formulation spontaneously forms 74.0 ± 1.7 nm sized nanovesicles with 72.9 ± 1.5 % entrapment efficiency. DSC and PXRD studies revealed that BU was present in phospholipid bilayer in amorphized form and TEM images confirmed the multi lamellar vesicular structure. Physicochemical stability of BU was significantly enhanced with proliposomal formulation. In-vivo studies in Sprague Dawley rats showed proliposomal formulation has comparable immunosuppression activity and 110.62 % relative bioavailability as compared to marketed Busulfan formulation i.e. Busulfex®.

Stability of busulfan solutions in polypropylene syringes and infusion bags as determined with an original assay.

PURPOSE: The stability of busulfan solution in 0.9% sodium chloride and stored in polypropylene syringes or infusion bags was evaluated. METHODS: Busulfan solutions (0.54 mg/mL) were prepared and transferred to 50-mL polypropylene syringes and 100- and 500-mL polypropylene infusion bags and stored at 2-8 and 23-27 °C. Chemical stability was measured using a stability-indicating, ultrahigh performance liquid chromatography coupled to mass spectrometry method. The stability of busulfan was assessed by measuring the percentage of the initial concentration remaining at the end of each time point of analysis. The initial busulfan concentration was defined as 100%. Stability was defined as retention of at least 90% of the initial busulfan concentration. A visual inspection of the samples for particulate matter, clarity, and color without instrumentation of magnification was conducted at each time point of analysis. RESULTS: The visual inspection demonstrated no influence of the storage container when busulfan infusions diluted in 0.9% sodium chloride injection were stored at 23-27 °C. No color change or precipitate was observed at this temperature; however, a rapid decrease of the busulfan content in all containers stored at room temperature was observed. Busulfan in syringes was chemically stable for 12 hours, while busulfan in infusion bags (100 and 500 mL) was stable only for 3 hours at 23-27 °C. CONCLUSION: Busulfan 0.54-mg/mL solution in 0.9% sodium chloride injection was physically and chemically stable for 30 hours when stored in 50-mL polypropylene syringes at 2-8 °C and protected from light.

Effect of Temperature on the Kinetics of the Activation of Treosulfan and Hydrolytic Decomposition of Its Active Epoxy Derivatives.

Treosulfan (TREO) is a prodrug applied in the treatment of ovarian cancer and a myeloablative conditioning prior to stem cell transplantation. A sequential activation of TREO to intermediate monoepoxide (S,S-EBDM) and then to (2S,3S)-1,2:3,4-diepoxybutane (S,S-DEB) involves a nonenzymatic intramolecular nucleophilic substitution. The aim of this study is to determine the effect of temperature on the rate constants (k) for the activation of TREO and the hydrolysis of its epoxy derivatives in a phosphate buffer of pH 7.4 at an ionic strength of 0.16-0.17 M. Over the tested temperature range, the ln of k changed linearly with a reciprocal of absolute temperature. The mean activation energy (Ea) values for the TREO → S,S-EBDM and S,S-EBDM → S,S-DEB reactions were close to each other (122 and 118 kJ/mol, respectively). In opposition, the Ea for the hydrolysis of S,S-EBDM and S,S-DEB differed significantly (140 and 80 kJ/mol, respectively), which indicates that the structure of S,S-EBDM hampers a nucleophilic attack of water on the epoxide ring. The obtained results show that a temperature change by 1°C, from 36.5°C to 37.5°C, entails a 17% increase in the k of TREO decay, which might lead to an increased TREO clearance in vivo.

Determination of prodrug treosulfan and its biologically active monoepoxide in rat plasma, liver, lungs, kidneys, muscle, and brain by HPLC-ESI-MS/MS method.

A prodrug treosulfan (TREO) is currently investigated in clinical trials for conditioning prior to hematopoietic stem cell transplantation. Bioanalysis of TREO and its active derivatives, monoepoxide (S,S-EBDM) and diepoxide, in plasma and urine underlay the pharmacokinetic studies of these compounds but cannot explain an organ pharmacological action or toxicity. Recently, distribution of TREO and S,S-EBDM into brain, cerebrospinal fluid, and aqueous humor of the eye has been investigated in animal models and the obtained results presented clinical relevance. In this paper, a selective and rapid HPLC-ESI-MS/MS method was elaborated and validated for the studies of disposition of TREO and S,S-EBDM in rat plasma, liver, lungs, kidneys, muscle, and brain. The two analytes and codeine, internal standard (IS), were isolated from 50µL of plasma and 100µL of supernatants of the tissues homogenates using ultrafiltration Amicon vials. Chromatographic resolution was accomplished on C18 column with isocratic elution. The limits of quantitation of TREO and S,S-EBDM in the studied matrices ranged from 0.11 to 0.93µM. The HPLC-MS/MS method was adequately precise and accurate within and between runs. The IS-normalized matrix effect differed among the tissues and was the most pronounced in a liver homogenate supernatant (approximately 0.55 for TREO and 0.35 for S,S-EBDM). Stability of the analytes in experimental samples was also established. The validated method for the first time enabled determination of TREO and S,S-EBDM in the six life-important tissues in rats following administration of the prodrug.

Characterization and intramolecular bonding patterns of busulfan: Experimental and quantum chemical approach.

The investigations of structural conformers, molecular interactions and vibrational characterization of pharmaceutical drug are helpful to understand their behaviour. In the present work, the 2D potential energy surface (PES) scan has been performed on the dihedral angles C6O4S1C5 and C25S22O19C16 to find the stable conformers of busulfan. In order to show the effects of long range interactions, the structures on the global minima of PES scan have been further optimized by B3LYP/6-311++G(d,p) method with and without empirical dispersion functional in Gaussian 09W package. The presence of n→σ* and σ→σ* interactions which lead to stability of the molecule have been predicted by natural bond orbital analysis. The strong and weak hydrogen bonds between the functional groups of busulfan were analyzed using quantum topological atoms in molecules analysis. In order to study the long-range forces, such as van der Waals interactions, steric effect in busulfan, the reduced density gradient as well as isosurface defining these interactions has been plotted using Multiwfn software. The spectroscopic characterization on the solid phase of busulfan has been studied by experimental FT-IR and FT-Raman spectra. From the 13C and 1H NMR spectra, the chemical shifts of individual C and H atoms of busulfan have been predicted. The maximum absorption wavelengths corresponding to the electronic transitions between the highest occupied molecular orbital and the lowest unoccupied molecular orbital of busulfan have been found by UV-vis spectrum.

Biodistribution of biodegradable polymeric nano-carriers loaded with busulphan and designed for multimodal imaging.

BACKGROUND: Multifunctional nanocarriers for controlled drug delivery, imaging of disease development and follow-up of treatment efficacy are promising novel tools for disease diagnosis and treatment. In the current investigation, we present a multifunctional theranostic nanocarrier system for anticancer drug delivery and molecular imaging. Superparamagnetic iron oxide nanoparticles (SPIONs) as an MRI contrast agent and busulphan as a model for lipophilic antineoplastic drugs were encapsulated into poly (ethylene glycol)-co-poly (caprolactone) (PEG-PCL) micelles via the emulsion-evaporation method, and PEG-PCL was labelled with VivoTag 680XL fluorochrome for in vivo fluorescence imaging. RESULTS: Busulphan entrapment efficiency was 83% while the drug release showed a sustained pattern over 10 h. SPION loaded-PEG-PCL micelles showed contrast enhancement in T 2 *-weighted MRI with high r 2* relaxivity. In vitro cellular uptake of PEG-PCL micelles labeled with fluorescein in J774A cells was found to be time-dependent. The maximum uptake was observed after 24 h of incubation. The biodistribution of PEG-PCL micelles functionalized with VivoTag 680XL was investigated in Balb/c mice over 48 h using in vivo fluorescence imaging. The results of real-time live imaging were then confirmed by ex vivo organ imaging and histological examination. Generally, PEG-PCL micelles were highly distributed into the lungs during the first 4 h post intravenous administration, then redistributed and accumulated in liver and spleen until 48 h post administration. No pathological impairment was found in the major organs studied. CONCLUSIONS: Thus, with loaded contrast agent and conjugated fluorochrome, PEG-PCL micelles as biodegradable and biocompatible nanocarriers are efficient multimodal imaging agents, offering high drug loading capacity, and sustained drug release. These might offer high treatment efficacy and real-time tracking of the drug delivery system in vivo, which is crucial for designing of an efficient drug delivery system.

The Myeloablative Drug Busulfan Converts Cysteine to Dehydroalanine and Lanthionine in Redoxins.

The myeloablative agent busulfan (1,4-butanediol dimethanesulfonate) is an old drug that is used routinely to eliminate cancerous bone marrow prior to hematopoietic stem cell transplant. The myeloablative activity and systemic toxicity of busulfan have been ascribed to its ability to cross-link DNA. In contrast, here we demonstrate that incubation of busulfan with the thiol redox proteins glutaredoxin or thioredoxin at pH 7.4 and 37 °C results in the formation of putative S-tetrahydrothiophenium adducts at their catalytic Cys residues, followed by ß-elimination to yield dehydroalanine. Both proteins contain a second Cys, in their catalytic C-X-X-C motif, which reacts with the dehydroalanine, the initial Cys adduct with busulfan, or the S-tetrahydrothiophenium, to form novel intramolecular cross-links. The reactivity of the dehydroalanine (DHA) formed is further demonstrated by adduction with glutathione to yield a lanthionine and by a novel reaction with the reducing agent tris(2-carboxyethyl)phosphine (TCEP), which yields a phosphine adduct via Michael addition to the DHA. Formation of a second quaternary organophosphonium salt via nucleophilic substitution with TCEP on the initial busulfan-protein adduct or on the THT(+)-Redoxin species is also observed. These results reveal a rich potential for reactions of busulfan with proteins in vitro, and likely in vivo. It is striking that several of the chemically altered protein products retain none of the atoms of busulfan, in contrast to typical drug-protein adducts or traditional protein modification reagents. In particular, the ability of a clinically used drug to convert Cys to dehydrolanine in intact proteins, and its subsequent reaction with biological thiols, is unprecedented.

Computational approaches to find the active binding sites of biological targets against busulfan.

Determination of electrophilic and nucleophilic sites of a molecule is the primary task to find the active sites of the lead molecule. In the present study, the active sites of busulfan have been predicted by molecular electrostatic potential surface and Fukui function analysis with the help of dispersion corrected density functional theory. Similarly, the identification of active binding sites of the proteins against lead compound plays a vital role in the field of drug discovery. Rigid and flexible molecular docking approaches are used for this purpose. For rigid docking, Hex 8.0.0 software employing fast Fourier transform (FFT) algorithm has been used. The partial flexible blind docking simulations have been performed with AutoDock 4.2 software; where a Lamarckian genetic algorithm is employed. The results showed that the most electrophilic atoms of busulfan bind with the targets. It is clear from the docking studies that busulfan has inhibition capability toward the targets 12CA and 1BZM. Graphical Abstract Docking of ligand and protein.