Pharmaceutical Research最新文献

Background: Thrombotic diseases remain a major global health burden. Current anticoagulants are often limited by bleeding risks and narrow therapeutic windows, largely due to their single-target mechanisms. In contrast, medicinal leeches secrete diverse peptides that naturally and synergistically modulate multiple steps of the hemostatic system.

Methods: This paper systematically reviews published biochemical, structural, functional, and omics studies on leech-derived anticoagulant peptides, classifying them according to their molecular targets and antithrombotic mechanisms.

Results: Leech-derived peptides act synergistically at multiple key points of the coagulation cascade: (1) they inhibit platelet adhesion and aggregation by blocking the vWF-collagen interaction or suppressing GPIIb/IIIa; (2) they directly inhibit core coagulation proteases, such as thrombin and factor Xa; and (3) they interfere with fibrin stabilization and promote its dissolution by inhibiting factor XIIIa or modulating the fibrinolytic and intrinsic protease systems. From a molecular perspective, multispecies omics analyses have revealed a significant expansion of antithrombotic gene families and identified numerous novel peptide candidate genes.

Conclusions: Leech-derived peptides provide a unique natural platform for multi-target anticoagulation and represent promising leads for next-generation antithrombotic agents. Combining traditional purification with genomics-guided discovery will accelerate mechanism elucidation, structural optimization, and translational development.

Objective: Lung cancer chemoprevention modalities are gaining wide attention as it is the second most diagnosed cancer type and the leading cause of cancer-related deaths. Our previous studies reported unique lung cancer chemoprevention capability with a repurposed drug combination of sulfasalazine (SAS) and disulfiram (DSF). However, their efficacy is limited by poor bioavailability. To overcome this challenge, we developed bioenhanced oil-in-water (o/w) nano self-emulsifying drug delivery system (Nano-SEDDS) formulations of SAS and DSF.

Methods: Unique isotropic Nano-SEDDS of SAS and DSF were developed and optimized using a single-step mix method followed by in vitro physicochemical characterization and stability studies. An in vivo pharmacokinetic and tissue-biodistribution study was undertaken to test the proposed hypothesis of bioavailability enhancement with Nano-SEDDS of SAS and DSF.

Results: The optimal Nano-SEDDS formulation exhibited low nanodroplet sizes (< 200 nm), high drug content, and 4.5-fold (p < 0.01) and 3.75-fold (p < 0.01) enhancement in in vitro dissolution of SAS and DSF compared to the respective free drugs. The Nano-SEDDS formulations were also confirmed to be stable at room temperature in compliance with ICH guidelines. Further, SAS Nano-SEDDS showed a dose-dependent increment in oral bioavailability as shown by a significant 7.9-fold (p < 0.0001) enhancement in dose-normalized AUC at a dose of 10 mg/kg compared to free drug treatment at a control dose of 250 mg/kg.

Conclusion: Overall, the studies corroborated the successful formulation of bioavailability-enhanced SAS and DSF Nano-SEDDS with future co-delivery applications for lung cancer prevention.

Objective: The purpose of this study is to analyze the impact of the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) transport characteristics of drugs on their intracerebral distribution in male cynomolgus monkeys.

Methods: Steady-state concentrations of 15 drugs (13 drugs and 2 compounds) transported by passive diffusion or via solute carrier (SLC) and/or ATP-binding cassette (ABC) transporters were measured in the frontal cortex interstitial fluid (ISF), lateral ventricular cerebrospinal fluid (CSF) and lumbar CSF of monkey brain by means of microdialysis and lumbar puncture methods. The values of brain ISF (or CSF)/plasma unbound concentration ratio (K_p,uu) were calculated to quantify the intracerebral distribution characteristics.

Results: The K_{p,uu,ISF,cortex} values of substrates of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)were much lower than unity. The ISF concentration of these drugs were increased by the co-administration of elacridar, suggesting active efflux of these drugs at the BBB. Contrary to expectations, some of P-gp substrates were efficiently distributed into the brain with K_{p,uu,ISF,cortex} > 2. The lateral ventricular CSF concentrations of drugs tended to be higher than the ISF concentrations, while the lumbar CSF concentrations were comparable to the ISF concentrations.

Conclusion: The results of this study suggest that the intracerebral distribution of the test drugs from the blood in the monkey brain should consider the contribution of influx transporters as well as efflux transporters. In addition, the lumbar CSF concentrations of the test drugs appear to be a useful surrogate marker of the ISF concentrations.

Background: While photostability testing conditions for biologics are often based on small molecule standards, the unique characteristics of proteins necessitate a deeper understanding of appropriate testing and controls. This study examines the effects of light stress on various presentations of somatropin, a therapeutic growth hormone.

Methods: Somatropin was exposed to light in lyophilized, reconstituted, and diluted forms. Quality attribute changes were analyzed using size exclusion chromatography, micro-fluidic imaging, imaged capillary isoelectric focusing, and liquid chromatography mass spectrometry (LC-MS).

Results: Light stress increased high molecular weight species (HMWS), particularly in liquid formulations, as shown by size exclusion chromatography (Lyophilized + 0.4%, Reconstituted + 2.7%, Diluted + 4.7%). Micro-fluidic imaging revealed no change in particle formation. All presentations exhibited shifts in charge variants, with increases in acidic species (Lyophilized + 2.8%, Reconstituted + 7.8%, Diluted + 6.2%) and basic (Lyophilized + 0.4%, Reconstituted + 0.7%, Diluted + 0.8%) . Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping detected increased methionine oxidation in light-exposed samples, correlating with higher protein concentration (M14- Lyophilized + 2.3%, Reconstituted + 7.4%, Diluted + 2.7%, M125- Lyophilized + 2.5%, Reconstituted + 2.9%). Diluted somatropin showed higher HMWS levels but reduced methionine-125 oxidation susceptibility compared to reconstituted formulations.

Conclusions: Light exposure altered product quality attributes, with more pronounced effects on liquid presentations. These findings provide insights into the distinct impacts of light exposure on different drug presentations throughout their lifecycle, highlighting the importance of tailored photostability testing for different product presentations of biologic drugs.

Objective: Polymeric microspheres (MS) have been developed with moderate success using aqueous emulsification (AqE) for small molecules and peptides with a few approved products. AqE faces a challenge to achieve optimal encapsulation of large hydrophilic molecules such as proteins/mAbs. To overcome this, a novel non-aqueous emulsification platform, "FluoriPack" (FP), was developed. FP is a solid/hydrocarbon/fluorocarbon (S/H/F) platform that enables high biologic loading within MS, with minimal impact on biologic integrity.

Methods: To develop MS using FP, a model protein was encapsulated in polyorthoester (POE) MS via non-aqueous emulsification. The continuous phase was Fluorinert™ FC-40 containing a fluorosurfactant (PicoSurf™ 1). Protein loaded MS were evaluated for morphology, PSD, % loading & in-vitro release (IVR), accelerated stability testing, and toxicity of blank microspheres. Encapsulated protein released from MS were evaluated for integrity and potency.

Results: Preliminary evaluation indicates that MS prepared via FP (FP-MS) were superior to AqE-MS, indicated by the surface morphology (non-porous versus porous), encapsulation (> 60% versus < 10%), and burst release (< 40% versus > 75%). FP-MS had a mean diameter of ~ 40 µm and were stable over 1-month at accelerated conditions. No toxicity was observed in mammalian cells with > 80% viability post FP-MS treatment. The biologic retained integrity post-encapsulation with minimal aggregation (Δ 1.1%) and high potency (> 80%). In vitro release evaluation revealed a sustained release of biologic over 9 days (~ 8% every 24 h).

Conclusion: MS prepared using FluoriPack achieved the desired quality attributes, enabling it to be a promising tool for sustained delivery of biologics.

Purpose: Amorphous active pharmaceutical ingredients (APIs) are generally considered to have significantly higher bioavailability, compared to their crystalline counterpart, due to the enhanced solubility of the disordered phase. However, an akin functionality can be also adopted by the particle size of the powdered API. In this case study, a detailed investigation of the particle-size-influenced properties of amorphous griseofulvin powders will be introduced.

Methods: The crystallization of amorphous griseofulvin powders in the range 20 - 1000 μm (+ 2 - 10 μm only for crystalline form) was studied calorimetrically, spectroscopically, and microscopically. Dissolution profiles of pharmaceutical tablets with incorporated either amorphous or crystalline griseofulvin were obtained under conditions simulating the path through the gastrointestinal tract.

Results: Standard crystal growth regime was accompanied by the rapid diffusionless growth mode, which was detected at low heating rates for the finest griseofulvin powders. The dissolution profiles of the pharmaceutical tablets with incorporated individual griseofulvin powder fractions were described in terms of the Korsmeyer-Peppas model (indicating the release by super case II transport).

Conclusion: Particle size was found to play dominant role in the dissolution kinetics, whereas the difference in the dissolution rates of the crystalline and amorphous particles was rather negligible. This is a beneficial finding, considering the very low stability of finely powdered amorphous griseofulvin, but at the same time, it negates the primary purpose of amorphization. Main benefit is thus that of the coarse amorphous griseofulvin powder, which can be utilized to fine-tune the dissolution profile due to its delayed dissolution.

Objective: Staphylococcus aureus is a microorganism that can behave as a commensal or as a life-threatening pathogen. Its remarkable capacity to acquire resistance to antimicrobial agents, particularly through mechanisms such as efflux pumps, places it among the major contributors to global morbidity and mortality. In this study, the acyclic diterpene phytol was investigated for its potential as an efflux pump inhibitor (EPI). Molecular docking was performed to evaluate its interaction with the QacA/B efflux pump, followed by in silico prediction of ADMET properties. In addition, the inhibitory effect of phytol on efflux activity was assessed in vitro against S. aureus.

Methods: To this end, the Minimum Inhibitory Concentration (MIC) was determined through serial microdilution in broth. The evaluation of efflux pump inhibition was assessed by measuring the reduction in MIC of ampicillin and ethidium bromide (EtBr) when combined with phytol at sub-inhibitory concentrations (MIC/8).

Results: Docking results showed that phytol has an average binding affinity of -4.7 kcal/mol with QacA, interacting with several amino acids at the binding site, additionally, the ADMET evaluation reveals Phytol as a promising compound for demonstrating low toxicological capacity.

Conclusions: However, despite the in silico interactions, the diterpene did not demonstrate direct antibacterial activity nor did it reduce the MIC of ampicillin or ethidium bromide, indicating that it does not function as an EPI.

Objective: The effectiveness of pressurized metered-dose inhalers (pMDIs) relies on correct inhalation technique. While prior studies investigated idealized breathing, the impact of real-life irregularities remains less understood. This study explores how real-life irregularities-pausing, coughing, and premature exhalation-alter aerosol transport and deposition in the airways.

Methods: Large-eddy simulations combined with a discrete phase model were performed on a realistic male airway geometry extending from the oral cavity to the fourth bronchial generation. Computational predictions were validated against in vitro experiments conducted under constant inhalation.

Results: Breathing irregularities substantially modified airflow dynamics and shifted deposition toward the upper airways. Coughing generated the strongest vortical structures and turbulence, followed by premature exhalation. Deposition in the left lung decreased from 19.9% during standard COPD inhalation to 2.1% during exhalation and 0.9% during coughing, while mouth-throat deposition increased to 35.2% during coughing compared to 14.5% under the COPD baseline condition. Exhalation caused higher overall particle loss (27.9%) than coughing (24.1%), but coughing produced more pronounced inertial impaction in the upper airways. Fine particles (< 2 µm) were largely exhaled (approximately 80%), whereas particles in the 2-5 µm range-considered optimal for deep lung delivery-were redirected and lost under disturbed flow conditions.

Conclusions: Irregular breathing patterns markedly decrease deep lung deposition and increase upper airway losses. Repeated puffs without adequate intervals may exacerbate this problem, leading to excessive upper-airway deposition and increasing the likelihood of side effects. These findings provide guidance for physicians to tailor puff number and timing, improving therapeutic efficacy while minimizing risks to patient safety.

Although India ranks among the largest producers and exporters of human vaccines, the delay in launch of newer therapeutics is larger than the average in G20 nations. This study aims to assess the stakeholders' perspective on the coherence, clarity, and operational impact of the New Drugs and Clinical Trial (NDCT) Rules 2019. The analysis revealed a nuanced appraisal of the NDCT Rules wherein three major clusters of challenges faced by pharmaceutical companies are regulatory, operational, and systemic. The regulatory barriers encompass prolonged approval timelines, ambiguous legal mechanisms, fragmented oversight across agencies. Operational factors include uneven distribution of trial sites, inadequate site capacity, ethics committee delays. Systemic challenges centred on non-uniform trial protocols, inconsistent documentation practices and absence of harmonized operational guidelines.

Purpose: Human choroid plexus papilloma (HIBCPP) cells derived from choroid plexus papilloma in the lateral ventricle are considered suitable model cells for the blood-cerebrospinal fluid barrier (BCSFB). Therefore, in this study, the transporters expressed in HIBCPP cells were identified, and the functions of representative transporters were evaluated.

Methods: Transporter protein and mRNA expression were analyzed in HIBCPP cells using quantitative proteomics and quantitative reverse transcription-PCR. Transporter functions were evaluated by cellular uptake and transcellular transport studies using typical substrates.

Results: The following solute carrier (SLC) and ATP-binding cassette (ABC) transporters involved in drug and nutrient transport were expressed in HIBCPP cells: glucose transporter 1 (GLUT1), monocarboxylate transporter 1 (MCT1), L-type amino acid transporter 1 (LAT1), cationic amino acid transporter 1 (CAT1), glutamate transporter (GLAST), SLC35F2, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP). Furthermore, the mRNA of organic anion/cation transporters, such as organic anion transporting polypeptide 1B3 (OATP1B3) and plasma membrane monoamine transporter (PMAT), were detected. Additionally, uptake of representative substrates of the SLC transporters LAT1, CAT1, GLAST, GLUT1, MCT1, and SLC35F2 in HIBCPP cells occurred in a time- and temperature-dependent manner and decreased in the presence of specific inhibitors. Furthermore, the representative substrates of these transporters were transported in a symmetric or asymmetric manner through HIBCPP cells between the apical (cerebrospinal fluid [CSF]) and basolateral (blood) sides.

Conclusion: Various nutrient and drug transporters are functionally expressed in HIBCPP cells. Therefore, HIBCPP cells could serve as a useful human BCSFB model to evaluate nutrient and drug transport between the CSF and blood.