Optimisation of enzyme-assisted extraction of camptothecin from Nothapodytes nimmoniana and its characterisation.

INTRODUCTION: Efficient extraction of camptothecin (CPT), an anticancer agent from the commercial source Nothapodytes nimmoniana (J. Graham) Mabb in India, is of paramount importance. CPT is present in the highest concentration in the stem portion, and the stem can be readily harvested without uprooting the plant. The fluorescence microscopy mapping of the bark matrix for CPT revealed its presence in a free form within both the outer (epidermal and cortical tissues) and inner (xylem and phloem tissues) sections. The bark matrix primarily consists of cellulose, hemicellulose, and lignin, rendering it woody, rigid, and resistant to efficient solvent penetration for CPT extraction. We proposed a hypothesis that subjecting it to disruption through treatment with hydrolytic enzymes like cellulase and xylanase could enhance solvent diffusion, thereby enabling a swift and effective extraction of CPT. OBJECTIVE: The present study was aimed at enzyme-assisted extraction, using cellulase and xylanase for hydrolytic disruption of the cells to readily access CPT from the stem of the plant N. nimmoniana (J. Graham) Mabb. METHODOLOGY: The hydrolytic cell disruption of ground powder from the stem bark was studied using cellulase and xylanase enzymes. The enzymatically pretreated stem bark powder was subsequently recovered by filtration, dried, and subjected to extraction with methanol to isolate CPT. This process was optimised through a Box-Behnken design, employing a one-factor-at-a-time approach to assess parameters such as enzyme concentration (2-10% w/w), pH (3-7), incubation time (6-24 h), and solid-to-solvent ratio (1:30-1:70 g/mL). CPT was characterised using proton nuclear magnetic resonance (1H-NMR) and Fourier transform infrared (FTIR) spectra, and a high-performance liquid chromatography (HPLC) method was developed for quantification. RESULTS: The cellulase and xylanase treatment resulted in the highest yields of 0.285% w/w and 0.343% w/w, with efficiencies of 67% and 81%, respectively, achieved in a significantly shorter time compared to the untreated material, which yielded 0.18% with an efficiency of only 42%. Extraction by utilising the predicted optimised process parameters, a nearly two-fold increase in the yield, was observed for xylanase, with incubation and solvent extraction times set at 16 and 2 h, respectively. Scanning electron microscopy (SEM) images of the spent material indicated perforations attributed to enzymatic action, suggesting that this could be a primary factor contributing to the enhanced extraction. CONCLUSION: Enzyme-mediated hydrolytic cell disruption could be a potential approach for efficient and rapid isolation of CPT from the bark of N. nimmoniana.

Experimentally Validated QSAR Model for Surface pK a Prediction of Heterolipids Having Potential as Delivery Materials for Nucleic Acid Therapeutics.

The application of lipid-based drug delivery technologies for bioavailability enhancement of drugs has led to many successful products in the market for clinical use. Recent studies on amine-containing heterolipid-based synthetic vectors for delivery of siRNA have witnessed the United States Food and Drug Administration (USFDA) approval of the first siRNA drug in the year 2018. The studies on various synthetic lipids investigated for delivery of such nucleic acid therapeutics have revealed that the surface pK a of the constructed nanoparticles plays an important role. The nanoparticles showing pK a values within the range of 6-7 have performed very well. The development of high-performing lipid vectors with structural diversity and falling within the desired surface pK a is by no means trivial and requires tedious trial and error efforts; therefore, a practical solution is called for. Herein, an attempt to is made provide a solution by predicting the statistically significant pK a through a predictive quantitative structure-activity relationship (QSAR) model. The QSAR model has been constructed using a series of 56 amine-containing heterolipids having measured pK a values as a data set and employing a partial least-squares regression coupled with stepwise (SW-PLSR) forward algorithm technique. The model was tested using statistical parameters such as r 2, q 2, and pred_r 2, and the model equation explains 97.2% (r 2 = 0.972) of the total variance in the training set and it has an internal (q 2) and an external (pred_r 2) predictive ability of ∼83 and ∼63%, respectively. The model was validated by synthesizing a series of designed heterolipids and comparing measured surface pK a values of their nanoparticle assembly using a 2-(p-toluidino)-6-napthalenesulfonic acid (TNS) assay. Predicted and measured surface pK a values of the synthesized heterolipids were in good agreement with a correlation coefficient of 93.3%, demonstrating the effectiveness of this QSAR model. Therefore, we foresee that our developed model would be useful as a tool to cut short tedious trial and error processes in designing new amine-containing heterolipid vectors for delivery of nucleic acid therapeutics, especially siRNA.

Novel bicephalous heterolipid based self-microemulsifying drug delivery system for solubility and bioavailability enhancement of efavirenz.

There is an increasing demand for new lipidic biocompatible and safe materials for self-microemulsifying drug delivery system (SMEDDS). The present work reports the synthesis, characterization, oral mucosal irritation study, and application of novel erucic acid ester of G0-PETIM dendron based bicephalous heterolipid (BHL) as an oil phase in SMEDDS using Efavirenz (EFA), a BCS class II drug with poor water solubility and poor bioavailability. Studies were conducted to optimize EFA SMEDDS using different ratios of the BHL as oil phase and surfactant: co-surfactant weight ratios (Km). At Km (1.5), the microemulsion was spontaneously formed in water with mean globule size of 22.78 ±â€¯0.25 nm and polydispersity index (PDI) of 0.23 ±â€¯0.031 with high drug loading efficiency of 80.35 ±â€¯3.1%. Standard stability tests were performed on EFA SMEDDS and the results indicated it to be highly stable. The in vitro dissolution profile of EFA SMEDDS showed >95% of the drug release within an hour and expectedly substantial enhancement in in vivo bioavailability was observed; almost 6-fold increase in bioavailability with parameters Cmax 5.2 µg/mL, Tmax 3 h, and AUC(0-∞) 23.48 µg/h/mL respectively as compared the plain suspension of the drug. In conclusion, the BHL can be used effectively as an oil phase in SMEDDS to enhance solubility and bioavailability of BCS Class II drugs. Further, it holds, in general, a great promise as a new excipient for solubility and bioavailability enhancements.

Antimicrobial cell penetrating peptides with bacterial cell specificity: pharmacophore modelling, quantitative structure activity relationship and molecular dynamics simulation.

Current research has shown cell-penetrating peptides and antimicrobial peptides (AMPs) as probable vectors for use in drug delivery and as novel antibiotics. It has been reported that the higher the therapeutic index (TI) the higher would be the bacterial cell penetrating ability. To the best of our knowledge, no in-silico study has been performed to determine bacterial cell specificity of the antimicrobial cell penetrating peptides (aCPP's) based on their TI. The aim of this study was to develop a quantitative structure activity relationship (QSAR) model, which can estimate antimicrobial potential and cell-penetrating ability of aCPPs against S. aureus, to confirm the relationship between the TI and aCPPs and to identify specific descriptors responsible for aCPPs penetrating ability. Molecular dynamics (MD) simulation was also performed to confirm the membrane insertion of the most active aCPPs obtained from the QSAR study. The most appropriate pharmacophore was identified to predict the aCPP's activity. The statistical results confirmed the validity of the model. The QSAR model was successful in identifying the optimal aCPP with high activity prediction and provided insights into the structural requirements to correlate their TI to cell penetrating ability. MD simulation of the best aCPP with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer confirmed its interaction with the membrane and the C-terminal residues of the aCPP played a key role in membrane penetration. The strategy of combining QSAR and molecular dynamics, allowed for optimal estimation of ligand-target interaction and confirmed the importance of Trp and Lys in interacting with the POPC bilayer. Communicated by Ramaswamy H. Sarma.

Fatty Acid Esters of G0-(Propyl Ether Imine) Dendron as Bicephalous Heterolipids for Permeation Enhancement in Transdermal Drug Delivery.

The objective of the present study was to synthesize, evaluate, and explore the potential of a new class of bicephalous heterolipids (BHLs) as chemical permeation enhancers (CPEs) with emphasis on investigation of the influence of variable chain lengths of BHLs with the G0-PETIM (poly(propyl ether imine)) dendron as a headgroup on skin permeation efficiency using diclofenac sodium (DS) as a model drug. These BHLs were synthesized, and chemical structures were confirmed by FT-IR, 1H NMR, 13C NMR, and ESI-MS. All BHLs were assessed for in vitro cytotoxicity and in vivo skin irritation studies that revealed their dermal safety. All respective DS gels with loaded BHLs were evaluated for skin permeation at varied concentrations, where gels with 1% concentration showed a significant increase in steady state flux (ERflux) as compared to the control gel and oleic acid loaded gel. The mechanism of enhanced skin permeation using BHL-loaded DS gels was examined using transepithelial electrical resistance (TEER), attenuated total reflection infrared (ATIR), and histomorphology, which showed significant reduction in the barrier integrity, alterations in the stratum corneum (SC), and compromising of the SC. These observations were concurrent with skin permeation results indicating that BHL enhancers could have wide scope as semisynthetic CPEs for transdermal drug delivery.

Rational approach for design and evaluation of anti-aggregation agents for protein stabilization: A case study of trehalose phenylalaninate.

The present work introduces new anti-aggregation agent (AAA) derived through our new approach for design and evaluation of anti-aggregation agent as a multi-purpose excipient to combat protein aggregation. Therapeutic proteins undergo aggregation due to even minor changes in environmental conditions like temperature, pH, shear and stress. Excipients play a vital role in prevention of aggregation. To stabilize a protein formulation different classes of excipients are used in combination after carefully selecting through laborious and time consuming trial and error experiments. To resolve these concerns, we have developed a rational approach based on molecular docking analysis and have designed, synthesized AAAs, and validated the approach by experimental studies. Trehalose phenylalaninate (TPA) has been synthesized and evaluated for stabilization of Bovine serum albumin (BSA). TPA was found to be non-toxic with a LC50 of >80µg/ml. BSA solutions with and without TPA were subjected to thermal and agitation stress and aggregation was monitored using sophisticated analytical techniques. The helical structure of BSA was completely retained in stressed samples at 0.1% concentration of TPA. SEC-HPLC clearly demonstrated the absence of aggregates in presence of TPA. Although aggregation was not seen in fluorescence spectra but quenching due to TPA was evident. Moreover, molecular dynamics study on BSA-TPA complex showed lower RMSD.

Ultrasound-assisted rapid extraction and kinetic modelling of influential factors: Extraction of camptothecin from Nothapodytes nimmoniana plant.

Ultrasound-assisted extraction (UAE) of commercially important natural product camptothecin (CPT) from Nothapodytes nimmoniana plant has been investigated. The influences of process factors such as electric acoustic intensity, solid to liquid ratio, duty cycle, temperature and particle size on the maximum extraction yield and kinetic mechanisms of the entire extraction process have been investigated. The kinetics results showed that increasing the intensity, duty cycle, solid to liquid ratio and decreasing the particle size lead to substantial increase in extraction yields compared to classical stirring extraction. Different kinetic models were applied to fit the experimental data. The second order rate model appears to be the best. The extraction rate constant, initial extraction rate and the equilibrium concentration for all experimental conditions have been calculated. SEM analysis of spent plant material clearly showed hollow openings on cell structure, which could be directly correlated to explosive disruption by the action of ultrasound waves. Overall 1.7-fold increase in extraction yields of CPT (0.32% w/w) and decrease in time from 6h to 18min was observed over the stirring method.

Trehalose Monooleate: A Potential Antiaggregation Agent for Stabilization of Proteins.

Protein aggregation is a major problem of therapeutic proteins because aggregation decreases their therapeutic activity and shelf life and induces immunogenicity. Stabilization against aggregation is commonly attained by addition of different excipients like sugars, surfactants, buffers, salts, amino acids, polymers, etc. Generally these excipients are required in combination for stabilization. Sugars are required at a higher concentration, and commonly used surfactants like polysorbates have shortcomings due to oxidative degradation. With a view to have a multipurpose excipient to be effective at a lower concentration, we designed antiaggregation agents (AAAs) that would encompass the functionalities of two or more conventional excipients and would curtail the number of excipients to be added for stabilization. Our first designed AAA, trehalose monooleate (TMO), is a sugar-fatty acid derivative. It has been evaluated in silico by docking on aggregation prone regions of model protein bovine serum albumin (BSA), and experimentally its effectiveness has been validated as stabilizer against agitation and thermal stress. TMO has a lower CMC of 6 mg/L, is nonhemolytic, and was found to be nontoxic by sulforhodamine B (SRB) colorimetric assay in Human Hepatoma Cell Line (Hep-G2) using adriamycin as positive contol. Various spectroscopic and separation analytical techniques were employed to monitor the aggregation profile of BSA in presence and absence of TMO. CD spectroscopy showed complete retention of helical structure at concentration as low as 0.05% of TMO, while fluorescence spectroscopy provided vital insights into conformational stability rendered by TMO. Native-PAGE and SEC-HPLC studies demonstrated absence of aggregates. Molecular dynamics study on BSA-TMO docked complex further substantiated the stabilization effect. Overall, it can be said that TMO has good antiaggregation property. The present work is a preliminary attempt toward understanding protein excipient interactions and chemistry to provide rational basis for designing a single excipient for stabilization of protein formulations.

Novel dendritic derivatives of unsaturated fatty acids as promising transdermal permeation enhancers for tenofovir.

This study was aimed at exploring the potential of unsaturated fatty acids (UFAs) [palmitoleic (PA), linoleic (LA), linolenic (LLA) and arachidonic acid (AA)], and their newly synthesized dendritic esters [PA1E, LA1E, LLA1E and AA1E] having basic tertiary nitrogen as the branching element as transdermal permeation enhancers for the delivery of tenofovir. The structures of the derivatives were confirmed by FTIR, NMR (1H and 13C) and HRMS. The in vitro cytotoxicity study revealed their biocompatibility. Amongst the UFAs, only PA and LLA exhibited transdermal enhancer potential [enhancement ratio (ER) of 1.35 and 2.9 respectively]. All synthesized derivatives at 1% w/w were found to be more effective enhancers as compared to their parent UFAs, with LLA1E being identified as the most superior (ER = 5.31). Further, the concentration effect study revealed that at 2% w/w LLA1E had a greater ER (6.11) as compared to its parent (ER = 3.85). The permeability data correlated with the observations made in the histomorphological and transepithelial electrical resistance (TEER) evaluations. There was no significant loss in the integrity of the epidermis, transcellular and intercellular route of transport across the epidermis, with drug and enhancer treatment having no permanent damage on the epidermis. The novel dendritic ester derivatives of the UFAs therefore can be considered as effective transdermal permeation enhancers.

Dendrimers - from organic synthesis to pharmaceutical applications: an update.

Dendrimers are a relatively new class of monodisperse polymers, which have tree-like spherical structures with well-defined sizes and shapes. Their unique structure has a significant impact on their physical and chemical properties. Research on dendrimers is of significant interest to scientists from all areas and their utility in various scientific fields, including pharmaceuticals, is expanding. The present review is comprehensive and covers different aspects of dendrimers viz. (1) synthesis, (2) properties and (3) pharmaceutical applications. The emphasis is on their applications as well as the current ongoing research status for drug targeting.

Aryl-free radical-mediated oxidative arylation of naphthoquinones using o-iodoxybenzoic acid and phenylhydrazines and its application toward the synthesis of benzocarbazoledione.

Oxidative arylation of naphthoquinones has been developed through combination of o-iodoxybenzoic acid with arylhydrazines under mild conditions at open atmosphere. Arylated naphthoquinones with different electronic properties were obtained in moderate to good yields. The postulated radical mediated mechanism is supported by radical trapping experiments. Developed protocol for direct arylation of naphthoquinones has been extended toward short, high yielding, and an effective synthesis of antitumor-antibiotic precursor such as benzocarbazoledione.

Oleodendrimers: a novel class of multicephalous heterolipids as chemical penetration enhancers for transdermal drug delivery.

This paper reports synthesis and evaluation of Janus type generation G-1 and G-2 dendrimers. The dendrimers have been constructed by linking two building blocks, dendrons and oleic acid, through ester and amide bonds and were well characterized by Fourier-transform infrared (FT-IR), (1)H NMR, (13)C NMR and electrospray ionization mass spectrometry (ESI-MS). The dendrimers have been evaluated for in vitro cytotoxicity using sulforhodamine B assay (SRB assay) and in vivo skin irritation potential. The ester linked dendrimers did not exhibit any cytotoxicity even up to 80 µg/ml while G-1 and G-2 generations dendrimers with amide linkage exhibited toxicity above 70 µg/ml and 21 µg/ml, respectively, none of the dendrimers showed any skin irritation. All the dendrimers, tested for their skin permeation enhancement potential using diclofenac sodium (DS) as a model drug at a concentration of 1% in gels, showed significant increase in steady-state flux (ERflux) of the drug as compared to control (without enhancer), and oleic acid. Amongst the dendrimers, the ester linked G-1 and G-2 dendrimers showed highest ERflux, 3.33 ± 0.31 and 3.39 ± 0.21, respectively.

A novel biocompatible bicephalous dianionic surfactant from oleic acid for solid lipid nanoparticles.

New biocompatible bicephalous dianionic surfactant with low CMC value was successfully synthesized and its efficacy in pharmaceutical drug delivery system was explored by preparing solid lipid nanoparticles (SLNs) of ketoconazole, a BCS class II drug. Oleic acid based dianionic surfactant was synthesized by attachment of oleic acid through ester linkage, to a bidentate head component, prepared by 1,4-addition of propanolamine and tert-butyl acrylate, followed by hydrolysis and neutralization with sodium bicarbonate. Its critical micelle concentration (CMC), hydrophilic-lipophilic balance and logPoctanol/water were found to be 1.6mM/L, 34.68 and -2.96, respectively. Cytotoxicity study, conducted using sulforhodamine B assay, showed LC50, GI50 and TGI values>80 µg/ml indicating its biocompatibility. It was found to be non-irritant by in vivo skin irritation study. The particle size, polydispersity index, and zeta potential of the optimized formulation were 98.2±1.74 nm, 0.462±0.016, and -49.6 mV, respectively. Further, the SLNs were characterized by scanning electron microscopy, differential scanning calorimetry and X-ray diffraction studies. Drug entrapment efficiency was found to be 87.42±0.31%, and the SLNs exhibited good stability over a period of 3 months. The current investigations, therefore, report the successful development of systematically optimized SLNs of ketoconazole.

Oleic acid based heterolipid synthesis, characterization and application in self-microemulsifying drug delivery system.

There is increasing demand for lipids owing to their use in formulating lipid based drug delivery systems of poorly soluble drugs. The present work discusses the synthesis, characterization of oleic acid based heterolipid and its use as oil in the development of self-microemulsifying drug delivery system (SMEDDS) for parenteral delivery. Synthesis was carried out by Michael addition of tert-butyl acrylate to 3-amino-1-propanol to obtain di-tert-butyl aminopropanol derivative. Reaction of this di-tert-butyl aminopropanol derivative with oleoyl chloride using p-dimethylaminopyridine as a coupling agent gave the desired heterolipid. It was characterized by (1)H NMR, (13)C NMR and MS to confirm the structure. It did not exhibit any measurable cytotoxicity, even up to 80µg/ml concentration. Application in parenteral drug delivery was explored using furosemide (FUR), a BCS class IV drug, as a model. FUR showed three times greater solubility in the heterolipid as compared to oleic acid. SMEDDSs were developed using heterolipid as oily phase, Solutol HS 15(®) as surfactant and ethanol as a co-surfactant. Developed SMEDDS could form spontaneous microemulsion on addition to various aqueous phases with mean globule size <70nm without any phase separation or drug precipitation even after 24h, and exhibited negligible hemolytic potential.

Regioselective oxidation of cholic acid and its 7ß epimer by using o-iodoxybenzoic acid.

Rational exploration directed by DFT (density functional theory) based atomic Fukui indices, lead to development of regioselective oxidation of cholic acid and its 7ß epimer by o-iodoxybenzoic acid. In case of cholic acid only, 7α-hydroxyl underwent oxidation, where as in its 7ß epimer the selectivity was towards 12α-hydroxy group. Since these oxidations are the key steps in synthesis of ursodeoxycholic acid starting from cholic acid these findings may be useful in devising a protection free synthetic route.

Oxidative conversion of alpha,alpha-disubstituted acetamides to corresponding one-carbon-shorter ketones using hypervalent iodine (lambda5) reagents in combination with tetraethylammonium bromide.

Alpha,alpha-disubstituted acetamides undergo oxidative dehomologation to give one-carbon-shorter ketones when reacted with a hypervalent iodine (lambda(5)) reagent in combination with tetraethylammonium bromide (TEAB) in various solvents. In further studies, one such combination of a hypervalent iodine (lambda(5)) reagent, o-iodoxybenzoic acid, and TEAB has been established as a new, mild, efficient, and general method for the transformation.

o-Iodoxybenzoic acid- and tetraethylammonium bromide-mediated oxidative transformation of primary carboxamides to one-carbon dehomologated nitriles.

A clean and efficient method for the oxidative transformations of primary carboxamides to one-carbon dehomologated nitriles using the combination of o-iodoxybenzoic acid and tetraethylammonium bromide has been developed. This method exhibits a broad scope and is expected to be of great utility in organic synthesis.

A mild, chemoselective oxidation of sulfides to sulfoxides using o-iodoxybenzoic acid and tetraethylammonium bromide as catalyst.

A mild, selective, and high-yielding method for oxidation of sulfides to sulfoxides using IBX and tetraethylammonium bromide in a variety of solvents is described. The method offers the advantage of short reaction times, no over-oxidation to sulfones, and compatibility to a wide range of functional groups.