Injectable Dendrimer Hydrogel Delivers Melphalan in Both Conjugated and Free Forms for Retinoblastoma.

We report the successful synthesis of an injectable dendrimer hydrogel (DH) carrying melphalan, a clinical drug for retinoblastoma treatment, in both conjugated and free forms. Polyamidoamine (PAMAM) dendrimer generation 5 (G5) is surface-modified with an acid-sensitive acetal-dibenzocyclooctyne linker and then undergoes azide-alkyne cycloaddition with melphalan-PEG-N3 conjugate to form G5-acetal-melphalan. During the DH gelation between G5-acetal-melphalan and PEG-diacrylate, free melphalan is added, resulting in a hydrogel (G5-acetal-melphalan-DH/melphalan) that carries the drug in both conjugated and free forms. Melphalan is slowly released from G5-acetal-melphalan-DH/melphalan, with the conjugated melphalan released more quickly at pH 5.3 due to acid-triggered acetal bond cleavage. The formulation's in vitro safety and efficacy were established on human corneal epithelia (HCE-2) and retinoblastoma cells (Y79). In an in vivo Y79 tumor xenograft model of retinoblastoma, intratumorally injected G5-melphalan-DH formulation prolonged tumor suppression. This injectable, multimodal, pH-responsive formulation shows promise for intravitreal injection to treat retinoblastoma.

Synthesis and In Vitro Activity of Novel Melphalan Analogs in Hematological Malignancy Cells.

Despite the continuous developments in pharmacology and the high therapeutic effect of new treatment options for patients with hematological malignancies, these diseases remain a major health issue. Our study aimed to synthesize, analyze in silico, and determine the biological properties of new melphalan derivatives. We obtained three methyl esters of melphalan having in their structures amidine moieties substituted with thiomorpholine (EM-T-MEL), indoline (EM-I-MEL), or 4-(4-morpholinyl) piperidine (EM-MORPIP-MEL). These have not yet been described in the literature. The in vitro anticancer properties of the analogs were determined against THP1, HL60, and RPMI8226 cells. Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (alkaline comet assay), and their ability to induce apoptosis (Hoechst33342/propidium iodide double staining method; phosphatidylserine translocation; and caspase 3/7, 8, and 9 activity measurements). Changes in mitochondrial membrane potential were examined using the specific fluorescence probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine). The EM-T-MEL derivative had the highest biological activity, showing higher cytotoxic and genotoxic properties than the parent drug. Moreover, it showed a high ability to induce apoptosis in the tested cancer cells. This compound also had a beneficial effect in peripheral blood mononuclear cells (PBMC). In conclusion, we verified and confirmed the hypothesis that chemical modifications of the melphalan structure improved its anticancer properties. The conducted study allowed the selection of the compound with the highest biological activity and provided a basis for chemical structure-biological activity analyses.

Exploration on the drug solubility enhancement in aqueous medium with the help of endo-functionalized molecular tubes: a computational approach.

One major problem in the pharmaceutical industry is the aqueous solubility of newly developed orally administered drug candidates. More than 50% of newly developed drug molecules suffer from low aqueous solubility. The therapeutic effects of drug molecules are majorly dependent on the bioavailability and, in essence, on the solubility of the used drug molecules. Thus, enhancement of drug solubility of sparingly soluble drug molecules is a need of modern times. Considering the high importance of drug solubility, we have computationally shown the enhancement of drug solubility for seven class II (poorly water-soluble) drug molecules in a water medium. The uses of supramolecular macrocycles have immense importance in the same field. Thus, we have used two synthetic supramolecular receptors named host-1a and host-1b to enhance the water solubility of fluorouracil, albendazole, camptothecin, clopidogrel, indomethacin, melphalan, and tolfenamic acid drug molecules. Biomedical engagements of a supramolecular receptor commence with the formation of stable host-drug complexes. These complexations enhance the water solubility of drug molecules and sustain the release rate and bioavailability of drug molecules. Thus, in this work, we focus on the formation of stable host-drug complexes in water medium. Molecular dynamics simulation is applied to analyze the structural features and the energetics involved in the host-drug complexation process. The information obtained at the atomistic level helps us gain better insights into the key interactions that operate to produce such highly stable complexes. Thus, we can propose that these two supramolecular receptors may be used as drug solubilizing agents, and patients will benefit from this theragnostic application shortly.

Evaluating the adverse effects of melphalan formulations.

INTRODUCTION: For multiple myeloma patients who respond to primary therapy, autologous hematopoietic stem cell transplant (HSCT) is considered standard of care with high-dose melphalan for transplant candidates. There are now two different melphalan formulations available, including a propylene glycol containing (PG-MEL) product and a propylene glycol-free (PG-free MEL) product. Although considered bioequivalent, there remains limited literature directly evaluating the adverse events between the two agents. We seek to assess the tolerability and severity of side effects between the two formulations in a real-life practice setting. METHODS: A retrospective, descriptive analysis was conducted of multiple myeloma patients who received autologous stem cell conditioning with either melphalan formulation when dosed at 100 mg/m2/dose for two consecutive doses. The primary outcome was the assessment of tolerability and severity of side effects. Tolerability was split into four major categories including hematologic toxicity, gastrointestinal toxicity, renal toxicity, and highest recorded mucositis grade. RESULTS: There were a total of 78 patients who received a melphalan preparation during the study. The median time to myeloablation and neutrophil engraftment was five and seven days post-HSCT, respectively, for all patients. Patients who received PG-free MEL were less likely to develop mucositis, with 22 (56%) reported highest grade 0, defined by World Health Organization oral toxicity scale, compared to those who received PG-MEL (33%), p = 0.04. CONCLUSION: There were minimal differences in tolerability or side effects observed between PG-free MEL and PG-MEL. These data may assist in better understanding the anticipated adverse effects of a high-dose melphalan conditioning therapy.

Solution stability of Captisol-stabilized melphalan (Evomela) versus Propylene glycol-based melphalan hydrochloride injection.

PURPOSE: The objective of this study was to compare the stability of recently approved Captisol-stabilized propylene glycol-free melphalan injection (Evomela™) against currently marketed propylene glycol-based melphalan injection. The products were compared as reconstituted solutions in vials as well as admixture solutions prepared from normal saline in infusion bags. METHODS: Evomela and propylene glycol-based melphalan injection were reconstituted in normal saline and organic custom diluent, respectively, according to their package insert instructions. The reconstituted solutions were diluted in normal saline to obtain drug admixture solutions at specific drug concentrations. Stability of the solutions was studied at room temperature by assay of melphalan and determination of melphalan-related impurities. RESULTS: Results show that based on the increase in total impurities in propylene glycol-based melphalan injection at 0.45 mg/mL, Evomela admixture solutions are about 5, 9, 15 and 29 times more stable at concentrations of 0.45, 1.0, 2.0 and 5.0 mg/mL, respectively. Results confirmed that reconstituted Evomela solution can be stored in the vial for up to 1 h at RT or for up to 24 h at refrigerated temperature (2-8 °C) with no significant degradation. After storage in the vial, it remains stable for an additional 3-29 h after preparation of admixture solution in infusion bags at concentrations of 0.25-5.0 mg/mL, respectively. In addition, Evomela solution in saline, at concentration of 5.0 mg/mL melphalan was bacteriostatic through 72 h storage at 2-8 °C. CONCLUSION: Formulation of melphalan with Captisol technology significantly improved stability compared to melphalan hydrochloride reconstituted with propylene-glycol based diluents.

Targeted Cancer Therapy Using Fusion Protein of TNFα and Tumor-Associated Fibronectin-Specific Aptide.

Tumor necrosis factor-α has shown potent antitumor effects in preclinical and clinical studies. However, severe side effects at less than therapeutic doses have limited its systemic delivery, prompting the need for a new strategy for targeted delivery of the protein to tumors. Here, we report a fusion protein of mouse tumor necrosis factor (TNF)-α (mTNFα) and a cancer-targeting, high-affinity aptide and investigate its therapeutic efficacy in tumor-bearing mice. A fusion protein consisting of mTNFα, a linker, and an aptide specific to extra domain B (EDB) of fibronectin (APTEDB), designated mTNFα-APTEDB, was successfully produced by expression in Escherichia coli. mTNFα-APTEDB retained specificity and affinity for its target, EDB. In mice bearing EDB-overexpressing fibrosarcomas, mTNFα-APTEDB showed greater efficacy in inhibiting tumor growth than mTNFα alone or mTNFα linked to a nonrelevant aptide, without causing an appreciable loss in body weight. Moreover, in vivo antitumor efficacy was further significantly increased by combination treatment with the chemotherapeutic drug, melphalan, suggesting a synergistic effect attributable to enhanced drug uptake into the tumor as a result of TNFα-mediated enhanced vascular permeability. These results suggest that a fusion protein of mTNFα with a cancer-targeting peptide could be a new anticancer therapeutic option for ensuring potent antitumor efficacy after systemic delivery.

Rapid and simple flow injection analysis tandem mass spectrometric method for the quantification of melphalan in a lipid-based drug delivery system.

RATIONALE: The use of the anticancer drug melphalan is limited due to its poor water solubility. To address this limitation, it is incorporated within a novel delivery system using ß-cyclodextrin-gemini surfactants (18:1ßCDg). METHODS: Herein, two fast and simple flow injection analysis/tandem mass spectrometric (FIA-MS/MS) methods are developed for the quantification of melphalan (Mel) within the drug delivery system so that the solubilization efficiency of the system can be assessed. FIA-MS/MS methods are developed using a triple quadrupole linear ion trap mass spectrometer, equipped with electrospray ionization (ESI) in the positive ion mode. A deuterated form of melphalan (melphalan-d8) was used as an internal standard (IS). The methods were validated according to the FDA guidance. RESULTS: A linearity in the range of 2-100 ng/mL and accuracy and precision below 15% were observed for all standard points and quality control samples. The intra- and inter-day variations and freeze-thaw stability were within the acceptable range according to the criteria set by regulatory guidelines. On the other hand, other stability measures, such as room temperature stability and long-term stability, did not meet the required guidelines in some cases, indicating the need for quick sample analysis upon preparation. Such a fact could have been overlooked if full method validation had not been performed. CONCLUSIONS: The developed methods were applied to determine the encapsulation/solubilization of the [18:1ßCDg/Mel] delivery system. 18:1ßCDg enhances the aqueous solubility of melphalan without the need for co-solvent. The highest melphalan solubility was observed at a melphalan18:1ßCDg/Mel complex molar ratio of 2:1. This study demonstrated that a fast analysis for the purpose of quantifying a chemically unstable drug, such as melphalan, is feasible and important for the development of commercial dosage forms.

Discovery of IDO1 and DNA dual targeting antitumor agents.

The development of small molecules for cancer immunotherapy is highly challenging and indoleamine 2,3-dioxygenase 1 (IDO1) represents a promising target. Inspired by the synergistic effects between IDO1 inhibitors and traditional antitumor chemotherapeutics, the first orally active dual IDO1 and DNA targeting agents were designed by the pharmacophore fusion strategy. The bifunctional hybrids exhibited enhanced IDO1 enzyme inhibitory activity and in vitro cytotoxicity as compared to IDO1 inhibitor 1-methyl-tryptophan and DNA alkylating agent melphalan. In a murine LLC tumor model, the dual targeting agents demonstrated excellent antitumor efficacy, highlighting the advantages of this novel design strategy to improve the efficacy of small molecule cancer immunotherapy.

Synthesis, 99m Tc-labeling, and preliminary biological evaluation of DTPA-melphalan conjugates.

Melphalan (MFL) is a typical nitrogen mustard for the treatment of many types of cancer. For the purpose to develop novel 99m Tc-labeled tumor imaging agents with SPECT, MFL was directly labeled by 99m Tc using diethylene triamine pentacetate acid (DTPA) as bifunctional chelating agent. The novel ligands were successfully synthesized by conjugation of DTPA to MFL to get monosubstituted DTPA-MFL and bis-substituted DTPA-2MFL. Radiolabeling was performed in high yield to get 99m Tc-DTPA-MFL and 99m Tc-DTPA-2MFL, respectively, which were hydrophilic and stable at room temperature. The high initial tumor uptake with retention, good tumor/muscle ratios, and satisfactory scintigraphic images suggested the potential of 99m Tc-DTPA-MFL and 99m Tc-DTPA-2MFL for tumor imaging. However, the slow normal tissue clearance would be a great obstacle. Further modification on the linker and/or 99m Tc-chelate to improve the tumor targeting efficacy and in vivo kinetic profiles is currently in progress.

Interactions of antitumour Sialyl Lewis X liposomes with vascular endothelial cells.

Recently, we showed that tetrasaccharide selectin ligand SiaLeX provided targeted delivery of liposomes loaded in the bilayer with melphalan lipophilic prodrug to tumour endothelium followed by severe injury of tumour vessels in a Lewis lung carcinoma model. Here, we study the impact of SiaLeX ligand on the interactions of liposomes with human umbilical vein endothelial cells (HUVEC) using flow cytometry, spectrofluorimetry and confocal microscopy. Liposomes composed of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol ester of melphalan, 8:1:1, by mol, and varying percentages of lipophilic SiaLeX conjugate were labelled with BODIPY-phosphatidylcholine. The increase in SiaLeX content in liposomes led to a proportional increase in their uptake by cytokine-activated cells as opposed to non-activated HUVEC: for 10% SiaLeX liposomes, binding avidity and overall accumulation increased 14- and 6-fold, respectively. The early stages of intracellular traffic of targeted liposomes in the activated cells were monitored by co-localisation with the trackers of organelles. Endocytosis of SiaLeX liposomes occurred mostly via clathrin-independent pathways, which does not contradict the available literature data on E-selectin localisation in the plasma membrane. Using dual fluorescence labelling, with rhodamine-labelled phospholipid and calcein encapsulated at self-quenching concentrations, we found that SiaLeX liposomes undergo rapid (within minutes) internalisation by activated HUVEC accompanied by the disruption of liposomes; non-activated cells consumed a negligible dose of liposomes during at least 1.5h. Our data evidence the selective effect of SiaLeX formulations on activated endothelial cells and indicate their potential for intracellular delivery of melphalan lipophilic prodrug.

Imaging the DNA Alkylator Melphalan by CEST MRI: An Advanced Approach to Theranostics.

Brain tumors are among the most lethal types of tumors. Therapeutic response variability and failure in patients have been attributed to several factors, including inadequate drug delivery to tumors due to the blood-brain barrier (BBB). Consequently, drug delivery strategies are being developed for the local and targeted delivery of drugs to brain tumors. These drug delivery strategies could benefit from new approaches to monitor the delivery of drugs to tumors. Here, we evaluated the feasibility of imaging 4-[bis(2-chloroethyl)amino]-l-phenylalanine (melphalan), a clinically used DNA alkylating agent, using chemical exchange saturation transfer magnetic resonance imaging (CEST MRI), for theranostic applications. We evaluated the physicochemical parameters that affect melphalan's CEST contrast and demonstrated the feasibility of imaging the unmodified drug by saturating its exchangeable amine protons. Melphalan generated a CEST signal despite its reactivity in an aqueous milieu. The maximum CEST signal was observed at pH 6.2. This CEST contrast trend was then used to monitor therapeutic responses to melphalan in vitro. Upon cell death, the decrease in cellular pH from ∼7.4 to ∼6.4 caused an amplification of the melphalan CEST signal. This is contrary to what has been reported for other CEST contrast agents used for imaging cell death, where a decrease in the cellular pH following cell death results in a decrease in the CEST signal. Ultimately, this method could be used to noninvasively monitor melphalan delivery to brain tumors and also to validate therapeutic responses to melphalan clinically.

Covalent adducts of melphalan with free amino acids and a model peptide studied by liquid chromatography/tandem mass spectrometry.

RATIONALE: Melphalan is a frequently used chemotherapeutical agent for the treatment of myeloma, breast cancer, ovarian cancer and sarcoma of soft tissue. A good knowledge of the reactivity of the drug toward the different amino acids, e.g. covalent adduct formation, is crucial for the understanding of its activity and side effects during cancer treatment. METHODS: The reactivity of melphalan and sites of adduct formation were studied by in vitro incubation of melphalan with free amino acids and glutathione as a model peptide. The formed covalent adducts were investigated using ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) using a triple-quadrupole instrument. Accurate mass measurements for the confirmation of characteristic product ions were performed on a quadrupole time-of-flight (QTOF) mass spectrometer. RESULTS: The incubation of melphalan with different classes of amino acids resulted in the formation of adducts on the amino and carboxyl termini, as well as adduct formation in the reactive side chains of Cys, Met, Tyr, His, Lys, Asp and Glu. All these melphalan adducts could be identified by their characteristic collision-induced dissociation (CID) product ion patterns. CONCLUSIONS: The present study demonstrates the reactivity of melphalan towards the functional groups of amino acids. The different alkylation site products show distinctive fragmentation patterns, which enable a fast identification of the different melphalan adducts. This study is a first important step towards a better understanding of the adduct formation in more complex molecules, e.g. peptides and proteins.

A Phase IIb, Multicenter, Open-Label, Safety, and Efficacy Study of High-Dose, Propylene Glycol-Free Melphalan Hydrochloride for Injection (EVOMELA) for Myeloablative Conditioning in Multiple Myeloma Patients Undergoing Autologous Transplantation.

Autologous stem cell transplantation (ASCT) after high-dose melphalan conditioning is considered a standard of care procedure for patients with multiple myeloma (MM). Current formulations of melphalan (eg, Alkeran for Injection [melphalan hydrochloride]; GlaxoSmithKline, Research Triangle Park, NC, USA) have marginal solubility and limited chemical stability upon reconstitution. Alkeran requires the use of propylene glycol as a co-solvent, which itself has been reported to cause such complications as metabolic/renal dysfunction and arrhythmias. EVOMELA (propylene glycol-free melphalan HCl; Spectrum Pharmaceuticals, Inc., Irvine, CA, USA) is a new i.v. melphalan formulation that incorporates Captisol (Ligand Pharmaceuticals, Inc., La Jolla, CA, USA), a specially modified cyclodextrin that improves the solubility and stability of melphalan and eliminates the need for propylene glycol. This new formulation has been shown to be bioequivalent to Alkeran. EVOMELA (200 mg/m(2)) was administered as 2 doses of 100 mg/m(2) each in a phase IIb, open-label, multicenter study to confirm its safety and efficacy as a high-dose conditioning regimen for patients with MM undergoing ASCT. At 5 centers, 61 patients (26 women) with a median age of 62 years (range, 32-73) were enrolled. All patients achieved myeloablation with a median time of 5 days post-ASCT, and all successfully achieved neutrophil and platelet engraftment with median times of 12 days post-ASCT and 13 days post-ASCT, respectively; treatment-related mortality on day 100 was 0%. Overall response rate (according to independent, blinded review) was high (100%), with an overall complete response rate of 21% (13% stringent complete response; 8% complete response) and overall partial response rate of 79% (61% very good partial response; 18% partial response). The incidence of grade 3 mucositis and stomatitis was low (10% and 5%, respectively) with no grade 4 mucositis or stomatitis reported (graded according to National Cancer Institute Common Terminology Criteria for Adverse Events). Based on investigators' assessment of mucositis using the World Health Organization (WHO) oral toxicity scale, 75% of patients had a shift in mucositis score from WHO grade 0 at baseline to a higher grade on study, of which 13% of patients reported WHO grade 3 as the worst post-treatment mucositis over the course of the study; there were no reports of WHO grade 4 mucositis during the study. This study confirms the efficacy and acceptable safety profile of EVOMELA, a new propylene glycol-free melphalan formulation, as a high-dose conditioning regimen for ASCT in patients with MM.

Efficacy of cryotherapy associated with laser therapy for decreasing severity of melphalan-induced oral mucositis during hematological stem-cell transplantation: a prospective clinical study.

Melphalan followed by hematopoietic stem-cell transplantation (HSCT) is the standard treatment for multiple myeloma and other hematopoietic neoplasms. However, high doses of melphalan cause severe oral mucositis (OM). The objective was to verify the efficacy of cryotherapy plus laser therapy on reduction of OM severity. HSCT patients undergoing melphalan chemotherapy (n = 71) were randomly divided into two groups according to OM treatment: oral cryotherapy performed with ice chips for 1 h 35 min followed by low-level laser therapy (InGaAIP, 660 nm, 40 mW, 6 J/cm(2) ) (n = 54) and laser therapy alone with the same protocol (n = 17). A control group (n = 33) was composed of HSCT patients treated with melphalan who received no specific treatment for OM. OM scores and clinical information were collected from D0 to D + 11. The cryotherapy/laser therapy group showed the lowest OM scores (maximum Grade I) and the lowest mean number of days (8 days) with OM in comparison with the other groups (p < 0.001). OM Grades III and IV were present with high frequency only in the control group. The association of cryotherapy with laser therapy was effective in reducing OM severity in HSCT patients who underwent melphalan conditioning.

A comparative study of chitosan and poloxamer based thermosensitive hydrogel for the delivery of PEGylated melphalan conjugates.

OBJECTIVE: Although the melphalan (ML) used extensively for the management of breast cancer, its clinical application is limited due to significant hemolytic activity. In the present work, a comparative analysis of two distinct in situ-based thermogelling polymers of PEGylated ML was performed. METHODS: Briefly, the PEGylated conjugate of the melphalan (MLPEG 5000) for local and sustained drug release action is loaded into two different thermogelling polymeric systems, namely chitosan- and poloxamer-based systems. The synthesized conjugate was loaded to a chitosan (MLP 5000) and poloxamer-based (MPX-CG) thermogelling injectable hydrogels. These thermogelling hydrogels were evaluated for in vitro hydrolysis, in vitro hemolytic activity. and in vitro anticancer activity. RESULTS: The lower percent cumulative hydrolysis was witness for both the hydrogels. MPX-CG and MLP 5000 hydrogels as predicted had shown lower percent cumulative hydrolysis of 3.31 ± 0.1 and 1.67 ± 0.1 after 6 h. The percentage hemolysis of MPX-CG and MLP 5000 even at a concentration of 32 µg/ml was found to be 39.23 ± 1.24% and 34.23 ± 2.24%, observed at 1 h, respectively. Both the hydrogels showed similar anticancer pattern, the MPX-CG hydrogel showed low cell viability of 8.4 ± 1.1% at a concentration of 150 µM and the MLP-5000 hydrogel showed slight higher cell viability (13.12 ± 5.4%) as compared with MPX-CG hydrogel. CONCLUSION: Hence, from the present study it can be well understood that both the chitosan- and the poloxamer-based thermogelling hydrogel proves to be an effective drug delivery systems for the delivery of the PEGylated conjugates.

Dipeptidyl peptidase IV as a potential target for selective prodrug activation and chemotherapeutic action in cancers.

The efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells. Recently, the enzyme dipeptidyl peptidase IV (DPPIV) was considered as a potential target for the delivery of chemotherapeutic drugs. The purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to DPPIV as a strategy to enhance their specificity. The expression profile of DPPIV was obtained for seven cancer cell lines using DNA microarray data from the DTP database, and was validated by RT-PCR. A prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline-glycine dipeptide moiety, followed by hydrolysis studies in the seven cell lines with a standard substrate, as well as the glycyl-prolyl-melphalan (GP-Mel). Lastly, cell proliferation studies were carried out to demonstrate enzyme-dependent activation of the candidate prodrug. The relative RT-PCR expression levels of DPPIV in the cancer cell lines exhibited linear correlation with U95Av2 Affymetrix data (r(2) = 0.94), and with specific activity of a standard substrate, glycine-proline-p-nitroanilide (r(2) = 0.96). The significantly higher antiproliferative activity of GP-Mel in Caco-2 cells (GI50 = 261 µM) compared to that in SK-MEL-5 cells (GI50 = 807 µM) was consistent with the 9-fold higher specific activity of the prodrug in Caco-2 cells (5.14 pmol/min/µg protein) compared to SK-MEL-5 cells (0.68 pmol/min/µg protein) and with DPPIV expression levels in these cells. Our results demonstrate the great potential to exploit DPPIV as a prodrug activating enzyme for efficient chemotherapeutic drug targeting.

Differential binding of plasma proteins by liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the bilayer.

Immediately upon contact with blood, nanosized drug delivery systems become coated with a so-called protein corona. The quantitative and qualitative composition of the corona defines not only the behavior of the nanocarrier in the circulation but, ultimately, the pharmacokinetics and biodistribution of the encapsulated drug as well. In turn, the composition of the protein corona depends on the surface properties of the nanoparticles, such as size and distribution of charge and functional groups on the particle surface. Liposomes belong to the most bio- and hemocompatible drug delivery systems feasible for intravenous route of administration required in chemotherapy of metastasizing tumors. However, knowledge on the interactions of liposomes of various compositions with blood plasma proteins remains fragmentary. Moreover, all nanosized drug delivery systems are potential targets for the innate immunity system, primarily the complement (C) system, which underlies frequent cases of hypersensitivity reactions. Recently, in a panel of in vitro hemocompatibility tests, we demonstrated that liposomes built of natural phospholipids - egg phosphatidylcholine and phosphatidylinositol from Saccharomyces cerevisiae - and loaded with diglyceride conjugates of anticancer drugs melphalan and methotrexate, did not affect the morphology and numbers of the main blood cell types. While preparations with melphalan prodrug were also inert in coagulation and C activation tests, methotrexate-loaded liposomes caused impaired coagulation and C activation. The aim of this work was to study the interactions of liposomes carrying prodrugs of melphalan and methotrexate with blood plasma proteins in vitro. Data on protein binding capacity of liposomes obtained with classical gel permeation chromatography techniques allowed for prediction of rather rapid elimination of the liposomes from circulation. A number of differences revealed through immunoblotting of the liposome-bound proteins agree with the previously obtained data on C activation. The possible mechanism of C activation by methotrexate-containing liposomes is discussed.

Investigation of Melphalan interaction as an alkylating agent with nucleotides by using surface enhanced Raman spectroscopy (SERS).

SERS (Surface Enhanced Raman Spectroscopy) is a new Raman spectroscopy which relies on Surface Plasmon Resonance (SPR) of metal nanoparticles. We have applied colloidal silver and gold nanoparticles as amplifier agents to enhance nucleotide Raman signals. It is observed that without these enhancing agents, it is impossible to investigate nucleotide spectrum due to weak Raman signals. Interaction mechanism of Melphalan, an anticancer drug with four nucleotides (Adenine, Cytosine, Guanine, Thymine) was investigated using SERS to detect and identify changes due to alkylating process in Raman spectra. After incubating Melphalan drug with nucleotides for 24 h at 37 °C, some changes occurred in SERS spectrum and interpretation of SERS spectra revealed the influence of the alkyl substitution on peaks and Raman shifts. After incubation of Melphalan with each nucleotide, intensity of relevant SERS signals assigned to Amid III group of Cytosine and Amid I of Thymine decreased significantly, confirming alkylating taking place. In this study, we also investigated the effect of nanoparticles type on nucleotide spectrum. We could not obtain useful information in the cases of guanine nucleotide. The SERS spectrum of Cytosine as an example of nucleotides in aqueous solution compared to solid state and results demonstrated that in solid state better signals were obtained than in liquid state.

Incrimination and impact on recovery times and effects of BN nanostructures on antineoplastic drug-electronic density study.

CONTEXT: By delivering the drug to the intended cell location, the use of nanomaterials in the drug delivery system may influence how the patient receives the medication and may assist in mitigating severe side effects. Density functional theory was used to assess the use of boron carbon nitride nanocages (BNCNCs), boron nitride (BNNSs), and boron carbon nitride nanosheets (BNCNSs) as melphalan (Mln) drug carriers in both the gaseous and fluid phases. We systematically examined the dipole moment, density of states, frontier molecular orbital, and optimal adsorption energy to understand the targeted drug delivery potential of these nanostructures. Adsorption energy analysis revealed that in both gas and water media, Mln drug adsorption takes place spontaneously on all the conjugated structures. The occurrence of adsorption energy as physisorbed energy suggests that the process is reversible, and desorption can take place with a much lower energy input. This physical contact is appropriate for the unquestionable unloading of Mln medications to the intended location. The reactivity is higher in BNNSs and BNCNSs, while the stability is higher in BNCNCs. The recovery time shows a shorter time for BNNSs and BNCNSs, while BNCNC shows a potential desorption time in higher temperature. These conclusions are corroborated by the results of the quantum theory of atoms in molecules (QTAIM). After the interaction analysis, it was observed that the BNCNCs can act as potential carriers for the melphalan. From dipole moment analysis, all three nanostructures show a high hydrophilic nature but quite higher in BNCNCs after doping in both media. Overall, all the structures show the potential carrier for melphalan drug. METHODS: The quantum mechanical approach, or DFT, has been used to study the fundamental structural, electrical, thermodynamic, and other aspects of proposed structures to develop an acceptable Mln drug detector. The adsorbate and all adsorbents were optimized via the hybrid B3LYP functional and the 6-311G + + (2d, p) basis set approach prior to the adsorption process. The Gaussian 09 package was used at 298 K as the constant temperature and 1 atm as the constant pressure. The structures are examined using the same functional models for solvation analysis-6-311 G + + (2d, p) and B3LYP-as well as the polarized continuum model (PCM) model as the foundation set. Density of states was studied using GaussSum 3.0 software. The interaction studies QTAIM and RDG were studied using VMD and Multiwfn software.

Analysis of novel melphalan hydrolysis products formed under isolated lung perfusion conditions using liquid chromatography/tandem mass spectrometry.

RATIONALE: Melphalan is a widely used cytotoxic agent in cancer treatments. This phenylalanine analog has been shown an effective drug in the treatment of breast cancer, multiple myeloma and melanoma of the extremities. A good knowledge of the drug's degradation and metabolism are crucial for understanding its activity during cancer treatments. METHODS: The formation of hydrolysis products of melphalan is studied using ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS). Aqueous melphalan solutions were incubated at elevated temperatures and analyzed by UPLC/MS/MS. Two previously described hydrolysis products, mono- and dihydroxymelphalan (MOH and DOH), were formed in vitro and could be characterized during MS/MS and high-resolution experiments. RESULTS: Novel compounds with m/z values >500 Da were discovered. Comparison of the fragmentation patterns of these new molecules with those of MOH and DOH show great similarities. The higher masses are explained by the presence of two or more melphalan units. In total, more than 15 new hydrolysis products were found. Experiments were set up to study the formation and the chemical structures of these molecules. CONCLUSIONS: The hydrolysis of melphalan is studied in the scope of a phase II clinical trial (isolated lung perfusion, ILuP). Patient samples were screened for the presence of all documented and novel melphalan hydrolysis products. This study reports the formation of a new class of oligomeric compounds in both in vivo and in vitro samples.