Molecular Pharmaceutics最新文献

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with insidious onset, rapid progression, and a very poor prognosis. CD47 is a transmembrane protein associated with the development and poor prognosis of pancreatic cancer. The aim of this study was to evaluate the diagnostic value of novel immunoPET tracers targeting CD47 in preclinical pancreatic cancer models. The association of CD47 expression with pancreatic cancer was analyzed using the Gene Expression Profiling Interactive Analysis platform. Immunohistochemical analysis of tissue microarrays was performed to detect CD47 expression in PDAC. CD47 expression levels on BxPC-3 and AsPC-1 cell membranes were compared using flow cytometry. A VHH (C2)-targeting human CD47 and its albumin-binding derivative (ABDC2) were labeled with ⁶⁸Ga or ⁸⁹Zr, respectively. The developed tracers were evaluated by immuno-positron emission tomography (immunoPET) imaging in tumor-bearing nude and CD47-humanized mice. [⁶⁸Ga]Ga-NOTA-C2 effectively detected tumor lesions in nude mice models and further showed confirmative imaging capacity in CD47-humanized PDAC models. Compared with [⁶⁸Ga]Ga-NOTA-C2, [⁸⁹Zr]Zr-DFO-ABDC2 had a significantly prolonged circulation time, increased tumor uptake, and reduced accumulation in the kidneys. Finally, biodistribution and histological staining confirmed the results of the immunoPET imaging studies. In this study, we validated that two novel VHH-derived molecular imaging tracers for immunoPET imaging ([⁶⁸Ga]Ga-NOTA-C2 and [⁸⁹Zr]Zr-DFO-ABDC2) can effectively annotate CD47 expression and diagnose PDAC in a target-specific manner. Clinical application of the imaging strategies may help select patients for CD47-targeted therapies and assess the response thereafter.

The bioavailability of poorly water-soluble active pharmaceutical ingredients (APIs) can be improved via the formulation of an amorphous solid dispersion (ASD), where the API is incorporated into a suitable polymeric carrier. Optimal carriers that exhibit good compatibility (i.e., solubility and miscibility) with given APIs are typically identified through experimental means, which are routinely labor- and cost-inefficient. Therefore, the perturbed-chain statistical associating fluid theory (PC-SAFT) equation of state, a popular thermodynamic model in pharmaceutical applications, is examined in terms of its performance regarding the computational pure prediction of API–polymer compatibility based on activity coefficients (API fusion properties were taken from experiments) without any binary interaction parameters fitted to API–polymer experimental data (that is, k_ij = 0 in all cases). This kind of prediction does not need any experimental binary information and has been underreported in the literature so far, as the routine modeling strategy used in the majority of the existing PC-SAFT applications to ASDs comprised the use of nonzero k_ij values. The predictive performance of PC-SAFT was systematically and thoroughly evaluated against reliable experimental data for almost 40 API–polymer combinations. We also examined the effect of different sets of PC-SAFT parameters for APIs on compatibility predictions. Quantitatively, the total average error calculated over all systems was approximately 50% in the weight fraction solubility of APIs in polymers, regardless of the specific API parametrization. The magnitude of the error for individual systems was found to vary significantly from one system to another. Interestingly, the poorest results were obtained for systems with self-associating polymers such as poly(vinyl alcohol). Such polymers can form intramolecular hydrogen bonds, which are not accounted for in the PC-SAFT variant routinely applied to ASDs (i.e., that used in this work). However, the qualitative ranking of polymers with respect to their compatibility with a given API was reasonably predicted in many cases. It was also predicted correctly that some polymers always have better compatibility with the APIs than others. Finally, possible future routes to improve the cost–performance ratio of PC-SAFT in terms of parametrization are discussed.

Nanoparticles (NPs) show great advantages in cancer treatment by enabling controlled and targeted delivery of payloads to tumor sites through the enhanced permeability and retention (EPR) effect. In this study, highly effective pH-responsive and biodegradable calcium orthophosphate@liposomes (CaP@Lip) NPs with a diameter of 110 ± 20 nm were designed and fabricated. CaP@Lip NPs loaded with hydrophobic paclitaxel and hydrophilic doxorubicin hydrochloride achieved excellent drug loading efficiencies of 70 and 90%, respectively. Under physiological conditions, the obtained NPs are negatively charged. However, they switched to positively charged when exposed to weak acidic environments by which internalization can be promoted. Furthermore, the CaP@Lip NPs exhibit an obvious structural collapse under acid conditions (pH 5.5), which confirms their excellent biodegradability. The “proton expansion” effect in endosomes and the pH-responsiveness of the NPs facilitate the release of encapsulated drugs from individual channels. The effectiveness and safety of the drug delivery systems were demonstrated through in vitro and in vivo experiments, with a 76% inhibition of tumor growth. These findings highlight the high targeting ability of the drug-loaded NPs to tumor sites through the EPR effect, effectively suppressing tumor growth and metastasis. By combining CaP NPs and liposomes, this study not only resolves the toxicity of CaP but also enhances the stability of liposomes. The CaP@Lip NPs developed in this study have significant implications for biomedical applications and inspire the development of intelligent and smart drug nanocarriers and release systems for clinical use.

The ability to bind plasma proteins helps in comprehending relevant aspects related to the pharmacological properties of many drugs. Despite the vital role of the drug mubritinib (MUB) in the prophylaxis of various diseases, its interaction with carrier proteins still needs to be clarified. The present work focuses on the interaction between MUB and Human serum albumin (HSA), investigated by employing multispectroscopic, biochemical, and molecular docking approaches. The results reveal that MUB has quenched HSA intrinsic fluorescence (following a static mechanism) by attaching very close (r = 6.76 ?) and with moderate affinity (K_b ≈ 10⁴ M^–1) to the protein site I (mainly by H-bonds, hydrophobic and Van der Waals forces). On one side, the HSA–MUB interaction has been accompanied by a slight disturbance in the HSA chemical environment (around the Trp residue) and protein secondary structure modifications. On another side, MUB competitively inhibits HSA esterase-like activity, which is very similar to other Tyrosine kinase inhibitors, and evidence that protein functional alterations have been triggered by MUB interaction. In summary, all of the presented observations can shed light on diverse pharmacological factors associated with drug administration.

Particles in biopharmaceutical products present high risks due to their detrimental impacts on product quality and safety. Identification and quantification of particles in drug products are important to understand particle formation mechanisms, which can help develop control strategies for particle formation during the formulation development and manufacturing process. However, existing analytical techniques such as microflow imaging and light obscuration measurement lack the sensitivity and resolution to detect particles with sizes smaller than 2 μm. More importantly, these techniques are not able to provide chemical information to determine particle composition. In this work, we overcome these challenges by applying the stimulated Raman scattering (SRS) microscopy technique to monitor the C–H Raman stretching modes of the proteinaceous particles and silicone oil droplets formed in the prefilled syringe barrel. By comparing the relative signal intensity and spectral features of each component, most particles can be classified as protein–silicone oil aggregates. We further show that morphological features are poor indicators of particle composition. Our method has the capability to quantify aggregation in protein therapeutics with chemical and spatial information in a label-free manner, potentially allowing high throughput screening or investigation of aggregation mechanisms.

Programmed cell death receptor 1 (PD-1) and its ligand PD-L1 are particularly interesting immune checkpoint proteins for human cancer treatment. Positron emission tomography (PET) imaging allows for the dynamic monitoring of PD-L1 status during tumor progression, thus informing patients’ response index. Herein, we report the synthesis of two linear peptide-based radiotracers, [⁶⁴Cu]/[⁶⁸Ga]HKP2201 and [⁶⁴Cu]/[⁶⁸Ga]HKP2202, and validate their utility for PD-L1 visualization in preclinical models. The precursor peptide HKP2201 was derived from a linear peptide ligand, CLP002, which was previously identified by phage display and showed nanomolar affinity toward PD-L1. Appropriate modification of CLP002 via PEGylation and DOTA conjugation yielded HKP2201. The dimerization of HKP2201 generated HKP2202. The ⁶⁴Cu and ⁶⁸Ga radiolabeling of both precursors was studied and optimized. PD-L1 expression in mouse melanoma cell line B16F10, mouse colon cancer cell line MC38, and their allografts were assayed by immunofluorescence and immunohistochemistry staining. Cellular uptake and binding assays were conducted in both cell lines. PET imaging and ex vivo biodistribution studies were employed in tumor mouse models bearing B16F10 and MC38 allografts. [⁶⁴Cu]/[⁶⁸Ga]HKP2201 and [⁶⁴Cu]/[⁶⁸Ga]HKP2202 were obtained with satisfactory radiocharacteristics. They all showed lower liver accumulation compared to [⁶⁴Cu]/[⁶⁸Ga]WL12. B16F10 and MC38 cells and their tumor allografts sections were verified to express PD-L1. These tracers demonstrated a concentration-dependent cell affinity and a comparable half-maximal effect concentration (EC₅₀) with radiolabeled WL12. Competitive binding and blocking studies demonstrated the specific target of these tracers to PD-L1. PET imaging and ex vivo biodistribution studies revealed notable tumor uptake in tumor-bearing mice and rapid clearance from blood and major organs. Importantly, [⁶⁴Cu]/[⁶⁸Ga]HKP2202 showed higher tumor uptake compared to [⁶⁴Cu]/[⁶⁸Ga]HKP2201. Of note, [⁶⁴Cu] labeled tracers showed longer retention in tumors than [⁶⁸Ga] labeled traces, indicating advantages in the long-term tracking of PD-L1 dynamics. In comparison, [⁶⁸Ga]HKP2201 and [⁶⁸Ga]HKP2202 showed lower liver accumulation, enabling its great potential in the fast detection of both primary and metastatic tumors, including hepatic carcinoma. [⁶⁴Cu]/[⁶⁸Ga]HKP2201 and [⁶⁴Cu]/[⁶⁸Ga]HKP2202 are promising PET tracers for visualizing PD-L1 status. Notably, their combination would cooperate in rapid diagnosis and subsequent treatment guidance. Future assessment of the radiotracers in patients is needed to fully evaluate their clinical value.

Lipid nanoparticle (LNP) delivery systems are widely used in the delivery of small-molecule drugs and nucleic acids. In this study, we prepared LNP-miR-155 by lipid nanomaterial technology and investigated the effects of LNP-miR-155 on β-catenin/transcription factor 4 (TCF4)/solute carrier family 31 member 1/copper transporter 1 (SLC31A1/CTR1) signaling and copper transport in colorectal cancer. For this, we used an LNP-miR-155 cy5 inhibitor and LNP-miR-155 cy5 mimics for the transfection of HT-29/SW480 cells. The transfection efficiency and uptake efficiency were detected by immunofluorescence. Relevant cell assays confirmed that the LNP-miR-155 cy5 inhibitor mediates the regulation of copper transport through the β-catenin/TCF4/SLC31A1 axis. The LNP-miR-155 cy5 inhibitor reduced cell proliferation, migration, and colony formation and promoted cell apoptosis. We also confirmed that miR-155 downregulates HMG box-containing protein 1 (HBP1) and adenomatous polyposis coli (APC) in cells and activates the function of β-catenin/TCF4 signaling. In addition, we found that the copper transporter, SLC31A1, is highly expressed in colorectal cancer cells. Furthermore, we also found that the complex β-catenin/TCF4 promotes the transcription of SLC31A1 by binding to its promoter region, which sustains the transport of copper from the extracellular region to the intracellular region and increases the activities of Cu²⁺-ATPase and superoxide dismutase (SOD). In summary, the LNP-miR-155 cy5 inhibitor regulates β-catenin/TCF4 by downregulating SLC31A1-mediated copper transport and intracellular copper homeostasis.

Idiopathic pulmonary fibrosis (IPF) causes worsening pulmonary function, and no effective treatment for the disease etiology is available now. Recombinant Human Relaxin-2 (RLX), a peptide agent with anti-remodeling and anti-fibrotic effects, is a promising biotherapeutic candidate for musculoskeletal fibrosis. However, due to its short circulating half-life, optimal efficacy requires continuous infusion or repeated injections. Here, we developed the porous microspheres loading RLX (RLX@PMs) and evaluated their therapeutic potential on IPF by aerosol inhalation. RLX@PMs have a large geometric diameter as RLX reservoirs for a long-term drug release, but smaller aerodynamic diameter due to their porous structures, which were beneficial for higher deposition in the deeper lungs. The results showed a prolonged release over 24 days, and the released drug maintained its peptide structure and activity. RLX@PMs protected mice from excessive collagen deposition, architectural distortion, and decreased compliance after a single inhalation administration in the bleomycin-induced pulmonary fibrosis model. Moreover, RLX@PMs showed better safety than frequent gavage administration of pirfenidone. We also found RLX-ameliorated human myofibroblast-induced collagen gel contraction and suppressed macrophage polarization to the M2 type, which may be the reason for reversing fibrosis. Hence, RLX@PMs represent a novel strategy for the treatment of IPF and suggest clinical translational potential.

In an earlier investigation, the critical cooling rate to prevent drug crystallization (CR_crit) during the preparation of nifedipine (NIF) amorphous solid dispersions (ASDs) was determined through a time–temperature transformation (TTT) diagram (Lalge et al. Mol. Pharmaceutics 2023, 20 (3), 1806–1817). The current study aims to use the TTT diagram to determine the critical cooling rate to prevent drug nucleation (CR_crit?N) during the preparation of ASDs. ASDs were prepared with each polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose acetate succinate (HPMCAS). The dispersions were first stored under conditions promoting nucleation and then heated to the temperature that favors crystallization. The crystallization onset time (t_C) was determined by differential scanning calorimetry and synchrotron X-ray diffractometry. TTT diagrams for nucleation were generated, which provided the critical nucleation temperature (50 °C) and the critical cooling rate to avoid nucleation (CR_crit?N). The strength of the drug–polymer interactions as well as the polymer concentration affected the CR_crit?N, with PVP having a stronger interaction than HPMCAS. The CR_crit of amorphous NIF was ～17.5 °C/min. The addition of a 20% w/w polymer resulted in CR_crit of ～0.05 and 0.2 °C/min and CR_crit?N of ～4.1 and 8.1 °C/min for the dispersions prepared with PVP and HPMCAS, respectively.