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The COVID-19 pandemic has raised concern regarding respiratory system diseases and oral inhalation stands out as an attractive non-invasive route of administration for pulmonary diseases such as chronic bronchitis, cystic fibrosis, COVID-19 and community-acquired pneumonia. In this context, we encapsulated azithromycin in polycaprolactone nanoparticles functionalized with phospholipids rich in dipalmitoylphosphatidylcholine and further produced a fine powder formulation by spray drying with monohydrated lactose. Nanoparticles obtained by the emulsion/solvent diffusion-evaporation technique exhibited a mean hydrodynamic diameter around 195-228 nm with a narrow monomodal size distribution (PdI < 0.2). Nanoparticle dispersions were spray-dried at different inlet temperatures, atomizing air-flow, aspirator air flow, and feed rate, using lactose as a drying aid, resulting in a maximal process yield of 63% and an encapsulation efficiency of 83%. Excipients and the dry powder formulations were characterized in terms of morphology, chemical structure, thermal analyses and particle size by SEM, FTIR, DSC/TGA and laser light diffraction. The results indicated spherical particles with 90% at 4.06 µm or below, an adequate size for pulmonary delivery. Aerosolization performance in a NGI confirmed good aerodynamic properties. Microbiological assays showed that the formulation preserves AZM antimicrobial effect against Staphylococcus aureus and Streptococcus pneumoniae strains, with halos above 18 mm. In addition, no formulation-related cytotoxicity was observed against the human cell lines BEAS-2B (lung epithelial), HUVEC (endothelial) and HFF1 (fibroblasts). Overall, the approach described here allows the production of AZM-PCL nanoparticles incorporated into inhalable microparticles, enabling more efficient pulmonary therapy of lung infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Madrid Sani, Ramos-Rocha, Sarcinelli, Chaves, Rocha, Léo, Cerize, Zanin, Feitosa and Rangel-Yagui.)

Acute lymphoblastic leukaemia is currently treated with bacterial L-asparaginase; however, its side effects raise the need for the development of improved and efficient novel enzymes. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice treated with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, through in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. In addition, our single mutants were highly stable in biological environment simulation, unlike the double-mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have a higher cytotoxic effect than WT on T-cell-derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological responses and to study the enzymes' pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C elicited lower physiological changes and immune/allergenic responses. In relation to the pharmacokinetic profile, S206C exhibited twofold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and fewer side effects.

Thermostability is an important and desired feature of therapeutic proteins and is critical for the success or failure of protein drugs development. It can be increased by PEGylation-binding of poly(ethylene glycol) moieties-or glycosylation-post-translational modification to add glycans. Here, the thermostability and thermodynamic parameters of native, PEGylated, and glycosylated versions of the antileukemic enzyme crisantaspase were investigated. First-order kinetics was found to describe the irreversible deactivation process. Activation energy of the enzyme-catalyzed reaction ( E *) was estimated for native, PEGylated, and glycosylated enzyme (10.2, 14.8, and 18.8 kJ mol -1 respectively). Half-life decreased progressively with increasing temperature, and longer half-life was observed for PEG-crisantaspase (87.74 min) at 50 °C compared to the native form (9.79 min). The activation energy of denaturation of PEG-crisantaspase (307.1 kJ mol -1 ) was higher than for crisantaspase (218.1 kJ mol -1 ) and Glyco-crisantaspase (120.0 kJ mol -1 ), which means that more energy is required to overcome the energy barrier of the unfolding process. According to our results, PEG-crisantaspase is more thermostable than its native form, while Glyco-crisantaspase is more thermosensitive.

Skin is the ultimate barrier between body and environment and prevents water loss and penetration of pathogens and toxins. Internal and external stressors, such as ultraviolet radiation (UVR), can damage skin integrity and lead to disorders. Therefore, skin health and skin ageing are important concerns and increased research from cosmetic and pharmaceutical sectors aims to improve skin conditions and provide new anti-ageing treatments. Biomolecules, compared to low molecular weight drugs and cosmetic ingredients, can offer high levels of specificity. Topically applied enzymes have been investigated to treat the adverse effects of sunlight, pollution and other external agents. Enzymes, with a diverse range of targets, present potential for dermatological use such as antioxidant enzymes, proteases and repairing enzymes. In this review, we discuss enzymes for dermatological applications and the challenges associated in this growing field., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋 𝑚𝑎𝑥 ) of 2.0 g L -1 was reached after 5 h of cultivation, corresponding to 𝑃 X  = 0.4 g L -1 h. The µ 𝑚𝑎𝑥 corresponded to 1.0 h -1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25 mg L -1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15 µg mL -1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase k M of 9.5 nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products., (© 2024 Wiley-VCH GmbH.)

L-asparaginase (ASNase) is an important biological drug used to treat Acute Lymphoblastic Leukemia (ALL). It catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream and, since ALL cells cannot synthesize Asn, protein synthesis is impaired leading to apoptosis. Despite its therapeutic importance, ASNase treatment is associated to side effects, mainly hypersensitivity and immunogenicity. Furthermore, degradation by plasma proteases and immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative to protect the enzyme from plasma proteases and enhance pharmacokinetics profile. In addition, PEGylation might prolong the in vivo circulation of liposomes owing to the spherical shielding conferred by the polyethylene (PEG) corona around the nanostructures. In this paper, ASNase was encapsulated in liposomal formulations composed by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing or not different concentrations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy (polyethylene glycol)-2000] (DSPE-PEG). Nanostructures of approximately 142-202 nm of diameter and polydispersity index (PDI) of 0.069 to 0.190 were obtained and the vesicular shape confirmed by Transmission Electron Microscopy (TEM and cryo-TEM). The encapsulation efficiency (%EE) varied from 10% to 16%. All formulations presented activity in contact with ASNase substrate, indicating the liposomes permeability to Asn and/or enzyme adsorption at the nanostructures' surface; the highest activity was observed for DMPC/DSPE-PEG 10%. Finally, we investigated the activity against the Molt 4 leukemic cell line and found a lower IC 50 for the DMPC/DSPE-PEG 10% formulation in comparison to the free enzyme, indicating our system could provide in vivo activity while protecting the enzyme from immune system recognition and proteases degradation.

Antimicrobial peptides (AMPs) are ubiquitous host defense peptides characterized by their antibiotic activity and lower propensity for developing resistance compared to classic antibiotics. While several AMPs have shown activity against antibiotic-sensitive and even multi-drug resistant strains, some bottlenecks to further development and clinical applications are still present, for instance, low antimicrobial activity, instability under physiological conditions, systemic toxicity and the potential for compromising the innate host defense immunity. Conjugation to molecules such as proteins, synthetic polymers, small molecules and nanoparticles are strategies under investigation to boost the therapeutic efficacy of AMPs. This review focuses on the design and application of AMPs' conjugates. In silico tools for creating new AMPs and AMPs' conjugates and their clinical development are also discussed. Furthermore, key future considerations regarding the major achievements and challenges of AMPs' conjugates in the antimicrobial resistance context are presented as a take-home message.

Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30-40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.

Doxorubicin (DOX) is a natural antibiotic with antineoplastic activity. It has been used for over 40 years and remains one of the most used drugs in chemotherapy for a variety of cancers. However, cardiotoxicity limits its use for long periods. To overcome this limitation, encapsulation in smart drug delivery systems (DDS) brings advantages in comparison with free drug administration (i.e., conventional anticancer drug therapy). In this review, we present the most relevant nanostructures used for DOX encapsulation over the last 10 years, such as liposomes, micelles and polymeric vesicles (i.e., polymersomes), micro/nanoemulsions, different types of polymeric nanoparticles and hydrogel nanoparticles, as well as novel approaches for DOX encapsulation. The studies highlighted here show these nanoformulations achieved higher solubility, improved tumor cytotoxicity, prolonged DOX release, as well as reduced side effects, among other interesting advantages., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Biological drugs or biopharmaceuticals off patent open a large market for biosimilars and biobetters, follow-on biologics. Biobetters, in particular, are new drugs designed from existing ones with improved properties such as higher selectivity, stability, half-life and/or lower toxicity/immunogenicity. Glycosylation is one of the most used strategies to improve biological drugs, nonetheless bioconjugation is an additional alternative and refers to the covalent attachment of polymers to biological drugs. Extensive research on novel polymers is underway, nonetheless PEGylation is still the best alternative with the longest clinical track record. Innovative trends based on genetic engineering techniques such as fusion proteins and PASylation are also promising. In this review, all these alternatives wereexplored as well as current market trends, legislation and future perspectives., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The metabolic function of catalase (CAT) is to prevent oxidative damage to tissues through the hydrolysis of hydrogen peroxide, which is a strong oxidizing agent. It has been suggested as an alternative to treat skin diseases related to oxidative stress, such as vitiligo. Owing to the instability associated to the protein nature, topical use of CAT is challenging and, in this sense, PEGylation can be an interesting alternative. Here, we conjugated CAT to methoxy-poly(ethylene oxide) (mPEG) of 10, 20 and 40 kDa, by means of a nucleophilic attack of ε-amino groups to an electron-deficient carbonyl group of the reactive PEG, resulting in site specifically PEGylated bioconjugates. PEGylation yields ranged from 31% ± 2% for CAT-PEG40 to 59% ± 4% for CAT-PEG20 and were strongly affected by the reaction pH owing to the protonation/deprotonation state of primary amines of lysine and N-terminal residues. PEGylated conjugates were purified by size-exclusion chromatography (purity > 95%) and characterized by circular dichroism. Irrespectively of MW, PEG did not affected CAT secondary and tertiary structure, but a decrease in specific activity was observed, more pronounced when PEGs of higher MWs were used. However, this loss of activity is compensated by the increased long-term stability, with a gain of >5 times in t 1/2 . In vitro antioxidant activity of CAT-PEG20 showed complete elimination of lipid peroxidation at the skin upper layer (stratum corneum) suitable for a topical use to treat vitiligo, as well as other skin conditions related to oxidative stress., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This work reports, for the encapsulation of l-asparaginase, an anticancer enzyme into hybrid PMPC 25 -PDPA 70 /PEO 16 -PBO 22 asymmetric polymersomes previously developed by our group, with loading capacities with over 800 molecules per vesicle. Enzyme-loaded polymersomes show permeability and capacity to hydrolyze l-asparagine, which is essential to cancer cells. The nanoreactors proposed in this work can be potentially used in further studies to develop novel therapeutic alternatives based on l-asparaginase.

Covalent attachment of therapeutic proteins to polyethylene glycol (PEG) is widely used for the improvement of its pharmacokinetic and pharmacological properties, as well as the reduction in reactogenicity and related side effects. This technique named PEGylation has been successfully employed in several approved drugs to treat various diseases, even cancer. Some methods have been developed to obtain PEGylated proteins, both in multiple protein sites or in a selected amino acid residue. This review focuses mainly on traditional and novel examples of chemical and enzymatic methods for site-selective PEGylation, emphasizing in N-terminal PEGylation, that make it possible to obtain products with a high degree of homogeneity and preserve bioactivity. In addition, the main assay methods that can be applied for the characterization of PEGylated molecules in complex biological samples are also summarized in this paper., (Copyright © 2019 Belén, Rangel-Yagui, Beltrán Lissabet, Effer, Lee-Estevez, Pessoa, Castillo and Farías.)

One of the main toxicities associated to intravenous administration of amphiphilic drugs is pronounced hemolytic activity. To overcome this limitation, we investigated the anti-hemolytic properties of polymeric micelles of Pluronics, triblock copolymers of poly(ethylene oxide) and poly(propylene oxide). We studied the encapsulation of the amphiphilic compound miltefosine (HePC) into polymeric micelles of Pluronics F108, F68, F127, L44, and L64. In vitro hemolysis indicated that, among the five copolymers studied, only F127 completely inhibited hemolytic effect of HePC at 50 μg/mL, this effect was also observed for other two amphiphilic molecules (cetyltrimethylammonium bromide and cethylpyridinium chloride). To better understand this interaction, we analyzed the HC 50 (concentration causing 50% of hemolysis) for HePC free and loaded into F127 micelles. Copolymer concentration influenced the hemolytic profile of encapsulated HePC; for F127 the HC 50 increased relative to free HePC (40 μg/mL) up to 184, 441, 736 and 964 μg/mL, for 1, 3, 6 and 9% F127, respectively. Interestingly, a linear relationship was found between HC 50 -HePC and F127 concentration. At 3% of F127, it is possible to load up to 300 μg/mL of HePC with no hemolytic effect. By achieving this level of hemolysis protection, a promising application is on the view, bringing the parenteral use of HePC and other amphiphilic drugs. Additionally, small-angle X-ray scattering (SAXS) was used to asses structural information on the interactions between HePC and F127 micelles., (Copyright © 2019 Elsevier B.V. All rights reserved.)

l-Asparaginase (ASNase) is an amidohydrolase used as a chemotherapeutic agent for the treatment of acute lymphoblastic leukemia (ALL). The nanoencapsulation of this enzyme is strategic to avoid its immediate immunogenic effects that lead to a decrease in the enzyme half-life. In this work, ASNase-containing nanoparticles (NPs) were prepared by double emulsification, through an ultrasonic sonicator or an Ultra-Turrax, using two copolymers of 50:50 (w/w) poly (lactic-co-glycolic acid) (PLGA) with different ranges of molecular weight (24-38 kDa and 30-60 kDa) and varying the concentration of polyvinyl alcohol (PVA) as a stabilizer (0.5, 1.0, 1.5 and 2.0%) as well as the emulsification time (30 and 60 s). Using 24-38 kDa PLGA and 1.0% PVA, we obtained by cavitation NPs with hydrodynamic diameter of 384 nm, polydispersity index of 0.143 and Zeta potential of -16.4 mV, whose ASNase encapsulation efficiency was as high as 87 ± 2%. The encapsulated enzyme showed an activity 22% higher than that of the free enzyme, and no conformational changes were detected by circular dichroism. The enzyme release from NPs entrapped in dialysis bags (500 kDa molecular weight cut-off) allowed selecting a controlled system able to release about 60% of the enzyme within 14 days, for which the Korsmeyer-Peppas model provided the best correlation (R 2  = 0.966)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Functional selectivity is a phenomenon observed in G protein-coupled receptors in which intermediate active-state conformations are stabilized by mutations or ligand binding, resulting in different sets of signaling pathways. Peptides capable of selectively activating β-arrestin, known as biased agonists, have already been characterized in vivo and could correspond to a new therapeutic approach for treatment of cardiovascular diseases. Despite the potential of biased agonism, the mechanism involved in selective signaling remains unclear. In this work, molecular dynamics simulations were employed to compare the conformational profile of the angiotensin II type 1 receptor (AT1R) crystal bound to angiotensin II, bound to the biased ligand TRV027, and in the apo form. Our results show that both ligands induce changes near the NPxxY motif in transmembrane domain 7 that are related to receptor activation. However, the biased ligand does not cause the rotamer toggle alternative positioning and displays an exclusive hydrogen-bonding pattern. Our work sheds light on the biased agonism mechanism and will help in the future design of novel biased agonists for AT1R.

L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

Polymersomes (PL), vesicles formed by self-assembly of amphiphilic block copolymers, have been described as promising nanosystems for drug delivery, especially of biomolecules. The film hydration method (FH) is widely used for PL preparation, however, it often requires long hydration times and commonly results in broad size distribution. In this work, we describe the challenges of the self-assembly of poly (ethylene glycol)-poly(lactic acid) (PEG-PLA) into PL by FH exploring different hydrophilic volume fraction ( f ) values of this copolymer, stirring times, temperatures and post-FH steps in an attempt to reduce broad size distribution of the nanostructures. We demonstrate that, alongside f value, the methods employed for hydration and post-film steps influence the PEG-PLA self-assembly into PL. With initial FH, we found high PDI values (>0.4). However, post-hydration centrifugation significantly reduced PDI to 0.280. Moreover, extrusion at higher concentrations resulted in further improvement of the monodispersity of the samples and narrow size distribution. For PL prepared at concentration of 0.1% ( m / v ), extrusion resulted in the narrower size distributions corresponding to PDI values of 0.345, 0.144 and 0.081 for PEG 45 -PLA 69 , PEG 114 -PLA 153 and PEG 114 -PLA 180 , respectively. Additionally, we demonstrated that copolymers with smaller f resulted in larger PL and, therefore, higher encapsulation efficiency (EE%) for proteins, since larger vesicles enclose larger aqueous volumes.

Miltefosine presents antineoplastic activity but high hemolytic potential. Its use in cancer has been limited to treating cutaneous metastasis of breast cancer. To decrease hemolytic potential, we developed a formulation of miltefosine-loaded polymeric micelles (PM) of the copolymer Pluronic-F127. A central composite design was applied and the analysis of variance showed that the optimum level of hydrodynamic diameter and polydispersity index predicted by the model and experimentally confirmed were 29nm and 0.105, respectively. Thermal analyses confirmed that miltefosine was molecularly dispersed within PM. Pluronic-F127 PM with miltefosine 80μM presented a significant reduction of hemolytic effect (80%, p<0.05) in comparison to free drug. In vitro assays against HeLa carcinoma cells demonstrated similar cytotoxicity to free miltefosine and PM. Our results suggest that, by lowering hemolytic potential, miltefosine-loaded Pluronic-F127 PM a promising alternative to broaden this drug use in cancer therapy, as well as of other alkylphosphocholines., (Copyright © 2017 Elsevier B.V. All rights reserved.)

l-asparaginase (l-asparagine amino hydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia and lymphosarcoma. It catalyzes l-asparagine (Asn) hydrolysis to l-aspartate and ammonia, and Asn effective depletion results in cytotoxicity to leukemic cells. Microbial l-asparaginase (ASNase) production has attracted considerable attention owing to its cost effectiveness and eco-friendliness. The focus of this review is to provide a thorough review on microbial ASNase production, with special emphasis to microbial producers, conditions of enzyme production, protein engineering, downstream processes, biochemical characteristics, enzyme stability, bioavailability, toxicity and allergy potential. Some issues are also highlighted that will have to be addressed to achieve better therapeutic results and less side effects of ASNase use in cancer treatment: (a) search for new sources of this enzyme to increase its availability as a drug; (b) production of new ASNases with improved pharmacodynamics, pharmacokinetics and toxicological profiles, and (c) improvement of ASNase production by recombinant microorganisms. In this regard, rational protein engineering, directed mutagenesis, metabolic flux analysis and optimization of purification protocols are expected to play a paramount role in the near future.

The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, β, and γ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The dopamine hypothesis states that decreased dopaminergic neurotransmission reduces schizophrenia symptoms. Neurokinin-3 receptor (NK3) antagonists reduce dopamine release and have shown positive effects in pre-clinical and clinical trials. We employed 2D and 3D-QSAR analysis on a series of 40 non-peptide NK3 antagonists. Multivariate statistical analysis, PCA and HCA, were performed to rational training/test set splitting and PLS regression was employed to construct all QSAR models. We constructed one highly predictive CoMFA model (q(2)= 0.810 and r(2)= 0.929) and acceptable HQSAR and CoMSIA models (HQSAR q(2)= 0.644 and r(2)= 0.910; CoMSIA q(2)= 0.691, r(2)= 0.911). The three different techniques provided convergent physicochemical results. All models indicate cyclopropane, piperidine and di-chloro-phenyl ring attached to cyclopropane ring and also the amide group attached to the piperidine ring could play an important role in ligand-receptor interactions. These findings may contribute to develop potential NK3 receptor antagonists for schizophrenia.

Coarse-grained molecular dynamics simulations are used to calculate the free energies of transfer of miltefosine, an alkylphosphocholine anticancer agent, from water to lipid bilayers to study its mechanism of interaction with biological membranes. We consider bilayers containing lipids with different degrees of unsaturation: dipalmitoylphosphatidylcholine (DPPC, saturated, containing 0%, 10%, and 30% cholesterol), dioleoylphosphatidylcholine (DOPC, diunsaturated), palmitoyloleoylphosphatidylcholine (POPC, monounsaturated), diarachidonoylphosphatidylcholine (DAPC, polyunsaturated), and dilinoleylphosphatidylcholine (DUPC, polyunsaturated). These free energies, calculated using umbrella sampling, were used to compute the partition coefficients (K) of miltefosine between water and the lipid bilayers. The K values for the bilayers relative to that of pure DPPC were found to be 5.3 (DOPC), 7.0 (POPC), 1.0 (DAPC), 2.2 (DUPC), 14.9 (10% cholesterol), and 76.2 (30% cholesterol). Additionally, we calculated the free energy of formation of miltefosine-cholesterol complexes by pulling the surfactant laterally in the DPPC + 30% cholesterol system. The free energy profile that we obtained provides further evidence that miltefosine tends to associate with cholesterol and has a propensity to partition into lipid rafts. We also quantified the kinetics of the transport of miltefosine through the various bilayers by computing permeance values. The highest permeance was observed in DUPC bilayers (2.28 × 10(-2) m/s) and the lowest permeance in the DPPC bilayer with 30% cholesterol (1.10 × 10(-7) m/s). Our simulation results show that miltefosine does indeed interact with lipid rafts, has a higher permeability in polyunsaturated, loosely organized bilayers, and has higher flip-flop rates in specific regions of cellular membranes.

Alkylphosphocholines (APCs) and alkyl-lysophosphocholines (ALPs) are antineoplastic agents that interfere with cellular membranes and signaling proteins. Protein kinase Cα (PKCα) is a signaling protein composed by catalytic (C3, C4) and regulatory domains (C1, C2). The C2 needs calcium (Ca(2+) ) and phosphatidylserine (PS) for activation. Miltefosine inhibits PKCα competitively with regard to PS and non-competitively with regard to Ca(2+) , however, the mechanism of action is unknown. We employed molecular docking, molecular dynamics and chemometric methods to verify how 7 APCs and ALPs derivatives and PS interact with the C2 domain. All ligands except PS were grouped in 2 clusters according to their interactions inside the enzyme. The findings showed that PS's phosphoryl oxygens interact with Ca(2+) , the serine moiety interacts with Asn189, and the carbonyl oxygen of the alkylic chain interacts with Arg249 and Thr251. On the other hand, ligands' phosphoryl oxygens interact with Asn189, Arg249, Thr250, and one water molecule instead of Ca(2+) . Because of the different binding mode, we hypothesize that the ligands cause conformational changes in the calcium binding region. Moreover, the packing mismatch between bilayer-forming lipids and ALP/APC chain impedes the C2 domain from docking to the internal leaflet of cellular membranes, interrupting PKCα activation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alkylphosphocholines (APC) are promising antitumor agents, which have the cellular membrane as primary target; however, red blood cell damage limits their wide therapeutic use. A variety of APC analogs has been synthesized and tested showing less hemolytic effect than the class prototype, Miltefosine (HePC). In this work, chemometric methods were applied to a set of 34 APC derivatives to identify the most relevant structural and molecular features of hemolytic activity. The APC derivatives were divided into three groups: (i) N-methylpiperidine and N-methylmorpholine derivatives with a long alkyl chain or flexible cyclopentadecyl rings, displaying a hemolytic rate of 17 %; (ii) adamantyl and cyclopentadecyl derivatives, showing an average hemolysis of 39 %; and, N,N,N-trimethylammonium, trans-N,N,N-trimethylcyclohexanamine, and trans-N,N,N-trimethylcyclopentanamine derivatives, whose average hemolysis was 41 %. The findings suggested that the presence of either bulky cationic head groups, or rings such as adamantyl and cyclohexyl, primarily increases the hemolysis of compounds with eleven atoms in the alkyl chain. Moreover, the macrocyclic cyclopentadecyl seems to be important to the hemolytic potential especially of compounds with five carbon atoms in the alkyl chain. Regarding linear carbon chain derivatives with no ring substitution, less bulky cationic head groups seem to favor hemolysis. Thus, in order to design more potent and less toxic APC antitumors, the reported structural/molecular patterns should not be included in their structure., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Since when alkylphospholipds (ALPs) were discovered and, even further after miltefosine's approval for the treatment of cutaneous metastasis of breast cancer and leishmaniasis, their activity against many other diseases have been extensively studied. This review aims to provide a summary of the alkylphospholipids' applications investigated so far., Results: The mechanism of action of ALPs is not fully understood, however it is believed that they interfere with lipid homeostasis leading to cell apoptosis. Due to ALPs cytotoxic activity, this class of molecules has shown to be effective against many diseases and conditions. Besides the approval of miltefosine for application in cutaneous metastasis of breast cancer and visceral and cutaneous leishmaniasis, several other analogs have proved efficacy and are promising as less toxic alternatives. ALPs have also shown in vitro and in vivo activity against Trypanosoma spp., amoebae, Tricomonas vaginalis, Schistosoma mansoni, HIV, and some fungi and bacteria species. The use of ALPs for intraocular lens coating is also under investigation. In addition, a clinical trial comparing miltefosine with usual treatments to spontaneous urticaria is also being conducted., Conclusions: Alkylphospholipids present such a broad pharmacological spectrum that justifies the need for further investigations of the drug class mechanisms of action, as well as the search for new analogs with improved activity and toxicological profiles.

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two-phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (K(GFPuv) < 1.00), and LPS removal into the micelle-rich phase (%REM(LPS) > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations., (Copyright © 2010 American Institute of Chemical Engineers (AIChE).)

Nitrofurazone (NF) and its derivative, hydroxymethylnitrofurazone (NFOH), have presented antichagasic activity. NFOH has higher activity and lower mutagenicity. The aim of this work was to assess whether NF and its derivative NFOH would also be inhibitors of cruzain, besides their trypanothione reductase inhibitory activity. In vitro cruzain inhibition tests were performed for both compounds, and the 50% inhibitory concentration (IC50) for NF and NFOH presented values of 22.83 +/- 1.2 microM and 10.55 +/- 0.81 microM, respectively. AM1 semi-empirical molecular modeling studies were performed to understand the activity of the compounds, corroborating the observed cruzain inhibitory activity.

Molecular modification is a quite promising strategy in the design and development of drug analogs with better bioavailability, higher intrinsic activity and less toxicity. In the search of new leads with potential antimicrobial activity, a new series of 14 4-substituted [N'-(benzofuroxan-5-yl)methylene]benzohydrazides, nifuroxazide derivatives, were synthesized and tested against standard and multidrug-resistant Staphylococcus aureus strains. The selection of the substituent groups was based on physicochemical properties, such as hydrophobicity and electronic effect. These properties were also evaluated through the lipophilic and electrostatic potential maps, respectively, considering the compounds with better biological profile. Twelve compounds exhibited similar bacteriostatic activity against standard and multidrug-resistant strains. The most active compound was the 4-CF(3) substituted derivative, which presented a minimum inhibitory concentration (MIC) value of 14.6-13.1 microg/mL, and a ClogP value of 1.87. The results highlight the benzofuroxan derivatives as potential leads for designing new future antimicrobial drug candidates.

The interaction of 5-nitro-2-furfurilylidene benzhydrazide (5NFB), potential anti-trypanosomal compound, with micellar solutions was studied. The results indicated that 50 mug of 5NFB completely kills 20 million T. cruzi epimastigote cells within 3 days, whereas the same amount of benznidazole kills 30% of the cells after 4 days. 5NFB solubility in surfactants solutions (SDS, DTAB, C12EO8) increased linearly with surfactant concentration. According to small angle X-ray scattering (SAXS), 5NFB does not affect micellar structural features. A comparison between C12EO8 effects on T. cruzi epimastigote cells and on erythrocytes showed that surfactant lytic effect is stronger in parasite cells, enlightening the potential of 5NFB micellar formulations.

Surfactants can be used to increase the solubility of poorly soluble drugs in water and to increase drug bioavailability. In this article, the aqueous solubilization of the nonsteroidal, antiinflammatory drug ibuprofen is studied experimentally and theoretically in micellar solutions of anionic (sodium dodecyl sulfate, SDS), cationic (dodecyltrimethylammonium bromide, DTAB), and nonionic (dodecyl octa(ethylene oxide), C12E8) surfactants possessing the same hydrocarbon "tail" length but differing in their hydrophilic headgroups. We find that, for these three surfactants, the aqueous solubility of ibuprofen increases linearly with increasing surfactant concentration. In particular, we observed a 16-fold increase in the solubility of ibuprofen relative to that in the aqueous buffer upon the addition of 80 mM DTAB and 80 mM C12E8 but only a 5.5-fold solubility increase upon the addition of 80 mM SDS. The highest value of the molar solubilization capacity (chi) was obtained for DTAB (chi = 0.97), followed by C12E8 (chi = 0.72) and finally by SDS (chi = 0.23). A recently developed computer simulation/molecular-thermodynamic modeling approach was extended to predict theoretically the solubilization behavior of the three ibuprofen/surfactant mixtures considered. In this modeling approach, molecular-dynamics (MD) simulations were used to identify which portions of ibuprofen are exposed to water (hydrated) in a micellar environment by simulating a single ibuprofen molecule at an oil/water interface (modeling the micelle core/water interface). On the basis of this input, molecular-thermodynamic modeling was then implemented to predict (i) the micellar composition as a function of surfactant concentration, (ii) the aqueous solubility of ibuprofen as a function of surfactant concentration, and (iii) the molar solubilization capacity (chi). Our theoretical results on the solubility of ibuprofen in aqueous SDS and C12E8 surfactant solutions are in good agreement with the experimental data. The ibuprofen solubility in aqueous DTAB solutions was somewhat overpredicted because of challenges associated with accurately modeling the strong electrostatic interactions between the anionic ibuprofen and the cationic DTAB. Our results indicate that computer simulations of ibuprofen at a flat oil/water interface can be used to obtain accurate information about the hydrated and the unhydrated portions of ibuprofen in a micellar environment. This information can then be used as input to a molecular-thermodynamic model of self-assembly to successfully predict the aqueous solubilization behavior of ibuprofen in the three surfactant systems studied.

Purpose: Micellar solubilization is a powerful alternative for dissolving hydrophobic drugs in aqueous environments. In this work, we provide an insight into this subject., Methods: A concise review of surfactants and micelles applications in pharmacy was carried out., Results: Initially, a description of surfactants and aqueous micellar systems is presented. Following, an extensive review on micellar drug solubilization, including both the principles involved on this phenomenon and the work already done regarding solubilization of drugs by micelles is presented. The application of micelles in drug delivery, in order to minimize drug degradation and loss, to prevent harmful side effects, and to increase drug bioavailability, is also presented. Special emphasis is given to the more recent use of polymeric micelles. Finally, we briefly discuss the importance of surfactants and micelles as biological systems models as well as its application in micellar catalysis., Conclusions: As can be seen from the review presented, the use of micelles in pharmacy is an important tool that finds numerous applications.

The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 445-456, 2003.)