In vitro gastrointestinal stability and intestinal absorption of ACE-1 and DPP4 inhibitory peptides from poultry by-product hydrolysates.

Bioactive peptides derived from food are promising health-promoting ingredients that can be used in functional foods and nutraceutical formulations. In addition to the potency towards the selected therapeutic target, the bioavailability of bioactive peptides is a major factor regarding clinical efficacy. We have previously shown that a low molecular weight peptide fraction (LMWPF) from poultry by-product hydrolysates possesses angiotensin-1-converting enzyme (ACE-1) and dipeptidyl-peptidase 4 (DPP4) inhibitory activities. The present study aimed to investigate the bioavailability of the bioactive peptides in the LMWPF. Prior to the investigation of bioavailability, a dipeptide YA was identified from this fraction as a dual inhibitor of ACE-1 and DPP4. Gastrointestinal (GI) stability and intestinal absorption of the bioactive peptides (i.e., YA as well as two previously reported bioactive dipeptides (VL and IY)) in the LMWPF were evaluated using the INFOGEST static in vitro digestion model and intestinal Caco-2 cell monolayer, respectively. Analysis of peptides after in vitro digestion confirmed that the dipeptides were resistant to the simulated GI conditions. After 4 hours of incubation, the concentration of the peptide from the apical side of the Caco-2 cell monolayer showed a significant decrease. However, the corresponding absorbed peptides were not detected on the basolateral side, suggesting that the peptides were not transported across the intestinal monolayer but rather taken up or metabolized by the Caco2 cells. Furthermore, when analyzing the gene expression of the Caco-2 cells upon peptide stimulation, a down-regulation of peptide transporters, the transcription factor CDX2, and the tight junction protein-1 (TJP1) was observed, suggesting the specific effects of the peptides on the Caco-2 cells. The study demonstrated that bioactive dipeptides found in the LMWPF were stable through in vitro GI digestion; however, the overall bioavailability may be hindered by inadequate uptake across the intestinal barrier.

Formulation of Saxagliptin Oral Films: Optimization, Physicochemical Characterization, In-Vivo Assessment, and In-Vitro Real-Time Release Monitoring via a Novel Polyaniline Nanoparticles-Based Solid-Contact Screen Printed Ion-Selective Electrode.

Oral dispersible films have received broad interest due to fast drug absorption and no first-path metabolism, leading to high bioavailability and better patient compliance. Saxagliptin (SXG) is an antidiabetic drug that undergoes first-path metabolism, resulting in a less active metabolite, so the development of SXG oral dispersible films (SXG-ODFs) improves SXG bioavailability. The formula optimisation included a response surface experimental design and the impact of three formulation factors, the type and concentration of polymer and plasticiser concentration on in-vitro disintegration time and folding endurance. Two optimised SXG-ODFs prepared using either polyvinyl alcohol (PVA) or hydroxypropyl methylcellulose were investigated. SXG-ODFs prepared with PVA demonstrated a superior rapid disintegration time, ranging from 17 to 890 s, with the fastest disintegration time recorded at 17 s. These short durations can be attributed to the hydrophilic nature of PVA, facilitating rapid hydration and disintegration upon contact with saliva. Additionally, PVA-based films displayed remarkable folding endurance, surpassing 200 folds without rupture, indicating flexibility and stability. The high tensile strength of PVA-based films further underscores their robust mechanical properties, with tensile strength values reaching up to 4.53 MPa. SXG exhibits a UV absorption wavelength of around 212 nm, posing challenges for traditional quantitative spectrophotometric analysis, so a polyaniline nanoparticles-based solid-contact screen-printed ion-selective electrode (SP-ISE) was employed for the determination of SXG release profile effectively in comparison to HPLC. SP-ISE showed a better real-time release profile of SXG-ODFs, and the optimised formula showed lower blood glucose levels than commercial tablets.

Fundamental evaluation regarding the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands.

OBJECTIVE: The marked success of prostate-specific membrane antigen (PSMA)-targeting radioligands with albumin binder (ALB) is attributed to the improvement of blood retention and tumor accumulation. [111In]In-PNT-DA1, our PSMA-targeting radioligand with ALB, also achieved improved tumor accumulation due to its prolonged blood retention. Although the advantage of ALBs is related to their reversible binding to albumin, the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands remains unclear because of the lack of information about radioligands with stronger albumin-binding than ALBs. In this study, we designed and synthesized [111In]In-PNT-DM-HSA, a new radioligand that consists of a PSMA-targeting radioligand covalently bound to albumin. The pharmacokinetics of [111In]In-PNT-DM-HSA was compared with those of [111In]In-PNT-DA1 and [111In]In-PSMA-617, a non-ALB-conjugated radioligand, to evaluate the relationship between albumin-binding and tumor accumulation. METHOD: The [111In]In-PNT-DM-HSA was prepared by incubation of [111In]In-PNT-DM, a PSMA-targeting radioligand including a maleimide group, and human serum albumin (HSA). The ability of [111In]In-PNT-DM-HSA was evaluated by in vitro assays. A biodistribution study using LNCaP tumor-bearing mice was conducted to compare the pharmacokinetics of [111In]In-PNT-DM-HSA, [111In]In-PNT-DA1, and [111In]In-PSMA-617. RESULTS: The [111In]In-PNT-DM-HSA was obtained at a favorable radiochemical yield and high radiochemical purity. In vitro assays revealed that [111In]In-PNT-DM-HSA had fundamental characteristics as a PSMA-targeting radioligand interacting with albumin covalently. In a biodistribution study, [111In]In-PNT-DM-HSA and [111In]In-PNT-DA1 showed higher blood retention than [111In]In-PSMA-617. On the other hand, the tumor accumulation of [111In]In-PNT-DA1 was much higher than [111In]In-PNT-DM-HSA and [111In]In-PSMA-617. CONCLUSIONS: These results indicate that the moderate reversible binding of ALB with albumin, not covalent binding, may play a critical role in enhancing the tumor accumulation of PSMA-targeting radioligands.

Development and first-in-human study of PSMA-targeted PET tracers with improved pharmacokinetic properties.

PURPOSE: A series of new 68Ga-labeled tracers based on [68Ga]Ga-PSMA-617 were developed to augment the tumor-to-kidney ratio and reduce the activity accumulation in bladder, ultimately minimize radiation toxicity to the urinary system. METHODS: We introduced quinoline group, phenylalanine and decanoic acid into different tracers to enhance their lipophilicity, strategically limiting their metabolic pathway through the urinary system. Their binding affinity onto LNCaP cells was determined through in vitro saturation assays and competition binding assays. In vivo metabolic study, PET imaging and biodistribution experiment were performed in LNCaP tumor-bearing B-NSG male mice. The most promising tracer was selected for first-in-human study. RESULTS: Four radiotracers were synthesized with radiochemical purity (RCP) > 95% and molar activity in a range of 20.0-25.5 GBq/µmol. The binding affinities (Ki) of TWS01, TWS02 to PSMA were in the low nanomolar range (< 10 nM), while TWS03 and TWS04 exhibited binding affinities with Ki > 20 nM (59.42 nM for TWS03 and 37.14 nM for TWS04). All radiotracers exhibited high stability in vivo except [68Ga]Ga-TWS03. Micro PET/CT imaging and biodistribution analysis revealed that [68Ga]Ga-TWS02 enabled clear tumor visualization in PET images at 1.5 h post-injection, with higher tumor-to-kidney ratio (T/K, 0.93) and tumor-to-muscle ratio (T/M, 107.62) compared with [68Ga]Ga-PSMA-617 (T/K: 0.39, T/M: 15.01) and [68Ga]Ga-PSMA-11 (T/K: 0.15, T/M: 24.00). In first-in-human study, [68Ga]Ga-TWS02 effectively detected PCa-associated lesions including primary and metastatic lesions, with lower accumulation in urinary system, suggesting that [68Ga]Ga-TWS02 might be applied in the detection of bladder invasion, with minimized radiation toxicity to the urinary system. CONCLUSION: Introduction of quinoline group, phenylalanine and decanoic acid into different tracers can modulate the binding affinity and pharmacokinetics of PSMA in vivo. [68Ga]Ga-TWS02 showed high binding affinity to PSMA, excellent pharmacokinetic properties and clear imaging of PCa-associated lesions, making it a promising radiotracer for the clinical diagnosis of PCa. Moreover, TWS02 with a chelator DOTA could also label 177Lu and 225Ac, which could be used for PCa treatment without significant side effects. TRIAL REGISTRATION: The clinical evaluation of this study was registered On October 30, 2021 at https://www.chictr.org.cn/ (No: ChiCTR2100052545).

Differential inhibition of sildenafil and macitentan on saxagliptin metabolism.

The development of diabetes mellitus (DM) is generally accompanied by erectile dysfunction (ED) and pulmonary arterial hypertension (PAH), which increases the use of combination drug therapy and the risk of drug-drug interactions. Saxagliptin for the treatment of DM, sildenafil for the treatment of ED and PAH, and macitentan for the treatment of PAH are all substrates of CYP3A4, which indicates their potential involvement in drug-drug interactions. Therefore, we investigated potential pharmacokinetic interactions between saxagliptin and sildenafil/macitentan. We investigated this speculation both in vitro and in vivo, and explored the underlying mechanism using in vitro hepatic metabolic models and molecular docking assays. The results showed that sildenafil substantially inhibited the metabolism of saxagliptin by occupying the catalytic site of CYP3A4 in a competitive manner, leading to the alterations in the pharmacokinetic properties of saxagliptin in terms of increased maximum plasma concentration (Cmax), area under the plasma concentration-time curve from time 0 to 24 h (AUC(0-t)), area under the plasma concentration-time curve from time 0 extrapolated to infinite time (AUC(0-∞)), decreased clearance rate (CLz/F), and prolonged terminal half-life (t1/2). In contrast, a slight inhibition was observed in saxagliptin metabolism when concomitantly used with macitentan, as no pharmacokinetic parameters were altered, except for CLz/F. Thus, dosage adjustment of saxagliptin may be required in combination with sildenafil to achieve safe therapeutic plasma concentrations and reduce the risk of potential toxicity, but it is not necessary for co-administration with macitentan.

Targeted interleukin-2 enhances the in vivo anti-cancer activity of Pluvicto™.

PURPOSE: Pluvicto™ ([177Lu]Lu-PSMA-617), a radioligand therapeutic targeting prostate-specific membrane antigen (PSMA), has been recently approved for the treatment of metastatic castration-resistant prostate cancer (mCRPR). The drug suffers from salivary gland and kidney uptake that prevents its dose escalation to potentially curative doses. In this work, we sought to potentiate the in vivo anti-cancer activity of Pluvicto™ by combining it with L19-IL2, a clinical-stage investigational medicinal product based on tumor-targeted interleukin-2. METHODS: We established a new PSMA-expressing model (HT-1080.hPSMA) and validated it using a fluoresceine analogue of PSMA-617 (compound 1). The HT-1080.hPSMA model was used to study the saturation and tumor retention of Pluvicto™ (compound 2) and to run combination therapy studies with L19-IL2. To complement our understanding of the mechanism of action of this novel combination, we conducted proteomics experiments on tumor samples after therapy with Pluvicto™ alone or in combination with the immunocytokine. RESULTS: High, selective, and long-lived tumor uptake was observed for Pluvicto™ (2) in the novel HT-1080.hPSMA model. Therapy studies in HT-1080.hPSMA tumor-bearing mice revealed that the combination of Pluvicto™ (2) plus L19-IL2 mediated curative and durable responses in all animals. Potent in vivo anti-cancer activity was observed solely for the combination modality, at doses that were well tolerated by treated animals. Proteomics studies indicated that L19-IL2 boosts the activation of the immune system in animals pre-treated with Pluvicto™. CONCLUSION: The therapeutic efficacy of Pluvicto™ at low radioactive doses can be effectively enhanced by the combination with L19-IL2. Our findings warrant further clinical exploration of this novel combination modality.

Physiologically Based Pharmacokinetic Modeling Characterizes the Drug-Drug Interaction Between Saxagliptin and Rifampicin in Patients With Renal Impairment.

The aim of the present study is to develop physiologically based pharmacokinetic (PBPK) models for saxagliptin and its active metabolite, 5-hydroxy saxagliptin, and to predict the effect of coadministration of rifampicin, a strong inducer of cytochrome P450 3A4 enzymes, on the pharmacokinetics of saxagliptin and 5-hydroxy saxagliptin in patients with renal impairment. The PBPK models of saxagliptin and 5-hydroxy saxagliptin were developed and validated in GastroPlus for healthy adults with or without rifampicin and adults with varying renal functions. Then, the effect of renal impairment combined with drug-drug interaction on saxagliptin and 5-hydroxy saxagliptin pharmacokinetics was investigated. The PBPK models successfully predicted the pharmacokinetics. For saxagliptin, the prediction suggests that rifampin greatly weakened the effect of renal impairment on reducing clearance, and the inductive effect of rifampin on parent drug metabolism seems to be increased with an increase in the degree of renal impairment severity. For patients with the same degree of renal impairment, rifampicin would have a slightly synergistic effect on the increase of 5-hydroxy saxagliptin exposure compared with dosed alone. There is an unsignificant decline for the saxagliptin total active moiety exposure values in patients with the same degree of renal impairment. It seems that patients with renal impairment are unlikely to require additional dose adjustments when coadministered with rifampicin, compared with saxagliptin alone. Our study provides a reasonable approach to explore unknown DDI potential in renal impairment.

Use of Physiologically Based Pharmacokinetic Modeling to Evaluate the Impact of Chronic Kidney Disease on CYP3A4-Mediated Metabolism of Saxagliptin.

We characterized the impact of chronic kidney disease (CKD) on the cytochrome P450 (CYP) 3A4-mediated metabolism of saxagliptin to its metabolite, 5-hydroxysaxagliptin, using a physiologically based pharmacokinetic (PBPK) model. A PBPK model of saxagliptin and its CYP3A4 metabolite, 5-hydroxysaxagliptin, was constructed and validated for oral doses ranging from 5 to 100 mg. The observed ratios of area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax ) between healthy subjects and subjects with CKD were compared with those predicted using PBPK model simulations. Simulations were performed with virtual CKD populations having decreased CYP3A4 activity (ie, 64%-75% of the healthy subjects' CYP3A4 abundance) and preserved CYP3A4 activity (ie, 100% of the healthy subjects' CYP3A4 abundance). We found that simulations using decreased CYP3A4 activity generally overpredicted the ratios of saxagliptin AUC and Cmax in CKD compared with those using preserved CYP3A4 activity. Similarly, simulations using decreased CYP3A4 activity underpredicted the ratio of 5-hydroxysaxagliptin AUC in moderate and severe CKD compared with simulations using preserved CYP3A4 activity. These findings suggest that decreased CYP3A4 activity in CKD underpredicts saxagliptin clearance compared with that observed clinically. Preserving CYP3A4 activity in CKD more closely estimates saxagliptin clearance and 5-hydroxysaxagliptin exposure changes observed in vivo. Our findings suggest that there is no clinically meaningful impact of CKD on the metabolism of saxagliptin by CYP3A4. Since saxagliptin is not a highly sensitive substrate and validated probe for CYP3A4, this work represents a case study of a CYP3A4 substrate-metabolite pair and is not a generalization for all CYP3A4 substrates.

Preparation of 177Lu-PSMA-617 in Hospital Radiopharmacy: Convenient Formulation of a Clinical Dose Using a Single-Vial Freeze-Dried PSMA-617 Kit Developed In-House.

OBJECTIVE: In the recent time, endoradionuclide therapy for metastatic castration-resistant prostate carcinoma employing 177Lu-PSMA-617 has yielded encouraging results and several clinical trials with the agent are currently ongoing. Routine preparation of 177Lu-PSMA-617 patient doses can be made simpler and convenient, if the ingredients essential for radiolabeling are made available in a ready-to-use lyophilized form. METHODS: PSMA-617 freeze-dried kit was formulated and used for the preparation of 177Lu-PSMA-617 clinical dose with high radiochemical purity using low/medium specific activity 177Lu. Detailed radiochemical studies were performed to determine the maximum activity and volume of 177LuCl3, which can be added in the kit for the formulation of 177Lu-PSMA-617. Studies were also performed to determine the shelf life of the kit to ensure its long-term usage. Studies were performed in buffer as well as human serum medium to determine the stability of the 177Lu-PSMA-617 complex after storing in respective media up to 7 days postpreparation. About ten patient doses of 177Lu-PSMA-617 were administered, and posttherapy scans were acquired. RESULTS: The formulated freeze-dried kit of PSMA-617 could be radiolabeled with an average percentage radiochemical purity > 98.53 ± 0.38. The freeze-dried kit was found suitable for tolerating up to 0.5 mL of 177LuCl3 (in 0.01 N HCl) and specific activity of 555 MBq/µg (15 mCi/µg) for the preparation of the patient dose of 177Lu-PSMA-617. The 177Lu-PSMA-617 complex prepared using the freeze-dried kit of PSMA-617 was observed to maintain % radiochemical purity (RCP) of 96.74 ± 0.87 and 94.81 ± 2.66, respectively, even after storing up to 7 days in buffer and human serum, respectively. 177Lu-PSMA-617 prepared using the in-house formulated freeze-dried kit of PSMA-617 exhibited accumulation in metastatic lesions picked up in a pretherapy PET scan. Reduction in number as well as size of lesions was observed in posttherapy scans acquired after two months of administering the first therapeutic dose of 177Lu-PSMA-617. CONCLUSIONS: The freeze-dried kit of PSMA-617 could be used for the preparation of 177Lu-PSMA-617 with high radiochemical purity (>98%) in a reproducible manner. 177Lu-PSMA-617 prepared using the developed kit was successfully evaluated in patients suffering from metastatic prostate cancer.

Formulation, Optimization, In Vitro and In Vivo Evaluation of Saxagliptin-Loaded Lipospheres for an Improved Pharmacokinetic Behavior.

The development and optimization of controlled release lipospheres (LS) from safe biocompatible behenic acid (BA) was performed for not only enhancing patient's compliance against highly prevailed chronic diabetes but also to vanquish the insufficiencies of traditional methods of drug delivery. The Box-Bhenken design (BBD) was utilized to statistically investigate the impact of formulation variables on percentage yield (Y 1), entrapment efficiency (Y 2), and SG-release (Y 3) from saxagliptin- (SG-) loaded LS, and the chosen optimized LS were subjected to a comparative in vivo pharmacokinetic analysis against commercially available SG brand. The compatibility analysis performed by DSC and FTIR established a complete lack of interaction of formulation components with SG, while p-XRD suggested a mild transformation of crystalline drug to its amorphous form during encapsulation process. The spherical, free flowing smooth surface LS having zeta potential of -32 mV and size range of 11-20 µm were conveniently formulated. The obtained data for Y 1 (30-80%), Y 2 (30-70%), and Y 3 (40-90%) showed a best fit with quadratic model. The pharmacokinetics analysis of LS showed a significantly decreased C max of SG (75.63 ± 3.85) with a sufficiently elevated T max (10.53 h) as compared to commercial brand of SG (99.66 ± 2.97 ng/mL and 3.55 ± 2.18 h). The achievement of greater bioavailability of SG was most probably attributed to higher level of half-life, mean residence time (MRT), and AUC0-24 for SG released from LS. Conclusively, the novel approach of SG-loaded LS had successfully sustained the plasma SG level for a prolonged time without increasing C max which would ultimately bring an effective management of chronic diabetes.

Evaluation of 177Lu and 47Sc Picaga-Linked, Prostate-Specific Membrane Antigen-Targeting Constructs for Their Radiotherapeutic Efficacy and Dosimetry.

Lu-177-based, targeted radiotherapeutics/endoradiotherapies are an emerging clinical tool for the management of various cancers. The chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) remains the workhorse for such applications but can limit apparent molar activity or efficient charge modulation, which can impact target binding and, as a consequence, target efficacy. Previously, our lab had developed the small, rare earth selective bifunctional chelator, picaga, as an efficient bifunctional chelator for scandium and lutetium isotopes. Here, we assess the performance of these constructs for therapy in prostate-specific membrane antigen (PSMA)-expressing tumor xenografts. To assess the viability of picaga conjugates in conjunction with long in vivo circulation, a picaga conjugate functionalized with a serum albumin binding moiety, 177Lu-picaga-Alb53-PSMA, was also synthesized. A directly comparative, low, single 3.7 MBq dose treatment study with Lu-PSMA-617 was conducted. Treatment with 177Lu-picaga-Alb53-PSMA resulted in tumor regression and lengthened median survival (54 days) when compared with the vehicle (16 days), 47Sc-picaga-DUPA-, 177Lu-picaga-DUPA-, and 177Lu-PSMA-617-treated cohorts (21, 23, and 21 days, respectively).

BCAAs and Di-Alanine supplementation in the prevention of skeletal muscle atrophy: preclinical evaluation in a murine model of hind limb unloading.

Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2 ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus - SOL - muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution.

Discovery and Characterisation of Highly Cooperative FAK-Degrading PROTACs.

Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.

Preclinical Assessment Addressing Intravenous Administration of a [68Ga]Ga-PSMA-617 Microemulsion: Acute In Vivo Toxicity, Tolerability, PET Imaging, and Biodistribution.

It has been herein presented that a microemulsion, known to be an effective and safe drug delivery system following intravenous administration, can be loaded with traces of [68Ga]Ga-PSMA-617 without losing its properties or causing toxicity. Following tolerated IV injections the capability of the microemulsion in altering [68Ga]Ga-PSMA-617 distribution was presented at 120 min post injection based on its ex vivo biodistribution results.

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 1: Exploration of Antibody Linker, Payload Loading, and Payload Molecular Properties.

The biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents. In this report, we describe the first phase of our systematic development of antibody-drug conjugates (ADCs) derived from bromodomain-containing protein 4 (BRD4)-targeting chimeric degrader entities. We demonstrate the antigen-dependent delivery of the degrader payloads to PC3-S1 prostate cancer cells along with related impacts on MYC transcription and intracellular BRD4 levels. These experiments culminate with the identification of one degrader conjugate, which exhibits antigen-dependent antiproliferation effects in LNCaP prostate cancer cells.

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 2: Improvement of In Vitro Antiproliferation Activity and In Vivo Antitumor Efficacy.

Heterobifunctional compounds that direct the ubiquitination of intracellular proteins in a targeted manner via co-opted ubiquitin ligases have enormous potential to transform the field of medicinal chemistry. These chimeric molecules, often termed proteolysis-targeting chimeras (PROTACs) in the chemical literature, enable the controlled degradation of specific proteins via their direction to the cellular proteasome. In this report, we describe the second phase of our research focused on exploring antibody-drug conjugates (ADCs), which incorporate BRD4-targeting chimeric degrader entities. We employ a new BRD4-binding fragment in the construction of the chimeric ADC payloads that is significantly more potent than the corresponding entity utilized in our initial studies. The resulting BRD4-degrader antibody conjugates exhibit potent and antigen-dependent BRD4 degradation and antiproliferation activities in cell-based experiments. Multiple ADCs bearing chimeric BRD4-degrader payloads also exhibit strong, antigen-dependent antitumor efficacy in mouse xenograft assessments that employ several different tumor models.

Discovery of First-in-Class Protein Arginine Methyltransferase 5 (PRMT5) Degraders.

The aberrant expression of protein arginine methyltransferase 5 (PRMT5) has been associated with multiple cancers. Using the proteolysis targeting chimera technology, we discovered a first-in-class PRMT5 degrader 15 (MS4322). Here, we report the design, synthesis, and characterization of compound 15 and two structurally similar controls 17 (MS4370) and 21 (MS4369), with impaired binding to the von Hippel-Lindau E3 ligase and PRMT5, respectively. Compound 15, but not 17 and 21, effectively reduced the PRMT5 protein level in MCF-7 cells. Our mechanism studies indicate that compound 15 degraded PRMT5 in an E3 ligase- and proteasome-dependent manner. Compound 15 also effectively reduced the PRMT5 protein level and inhibited growth in multiple cancer cell lines. Moreover, compound 15 was highly selective for PRMT5 in a global proteomic study and exhibited good plasma exposure in mice. Collectively, compound 15 and its two controls 17 and 21 are valuable chemical tools for exploring the PRMT5 functions in health and disease.

SUPPORT-1 (Subjects Undergoing PCI and Perioperative Reperfusion Treatment): A Prospective, Randomized Trial of CMX-2043 in Patients Undergoing Elective Percutaneous Coronary Intervention.

OBJECTIVE: The natural molecule α-lipoic acid has been shown to be partially cytoprotective through antioxidant and antiapoptotic mechanisms. To obtain an initial assessment of the safety and potential efficacy of a synthetic derivative, CMX-2043, in preventing ischemic complications of percutaneous coronary intervention (PCI) we conducted the Subjects Undergoing PCI and Perioperative Reperfusion Treatment (SUPPORT-1) trial, the first patient experience with this agent. METHODS AND RESULTS: SUPPORT-1 was a phase 2a, 6-center, international, placebo-controlled, randomized, double-blind trial. A total of 142 patients were randomized to receive a single intravenous bolus dose of drug or placebo administered 15-60 minutes before PCI. Cardiac biomarker assessments included serial measurements of creatine kinase myocardial band (CK-MB) at 6, 12, 18, and 24 hours after PCI and a single measurement of troponin T (TnT) at 24 hours. Peak concentrations of CK-MB and TnT were significantly reduced in the 2.4 mg/kg group compared with placebo (P = 0.05 and 0.03, respectively). No subject administered 2.4 mg/kg of CMX-2043 had an increase of CK-MB to ≥3X upper limit of normal versus 16% for placebo (P = 0.02); 16% of the 2.4-mg/kg dose group developed an elevation of TnT to ≥3X upper limit of normal versus 39% in the placebo group (P = 0.05). No drug-related serious adverse events were observed in any group. CONCLUSION: These data suggest that CMX-2043 may reduce PCI periprocedural myonecrosis and support further clinical evaluation of this novel agent for its potential cytoprotective effects.

Pharmacokinetic functions of human induced pluripotent stem cell-derived small intestinal epithelial cells.

To develop a novel intestinal drug absorption system using intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells, the cells must possess sufficient pharmacokinetic functions. However, the CYP3A4/5 activities of human iPS cell-derived small intestinal epithelial cells prepared using conventional differentiation methods is low. Further, studies of the CYP3A4/5 activities of human iPS-derived and primary small intestinal cells are not available. To fill this gap in our knowledge, here we used forskolin to develop a new differentiation protocol that activates adenosine monophosphate signaling. mRNA expressions of human iPS cell-derived small intestinal epithelial cells, such as small intestine markers, drug-metabolizing enzymes, and drug transporters, were comparable to or greater than those of the adult small intestine. The activities of CYP3A4/5 in the differentiated cells were equal to those of human primary small intestinal cells. The differentiated cells had P-glycoprotein and PEPT1 activities equivalent to those of Caco-2 cells. Differentiated cells were superior to Caco-2 cells for predicting the membrane permeability of drugs that were absorbed through a paracellular pathway and via drug transporters. In summary, here we produced human iPS cell-derived small intestinal epithelial cells with CYP3A4/5 activities equivalent to those of human primary small intestinal cells.

Lipospheres for Simultaneous Controlled Release and Improved Pharmacokinetic Profiles of Saxagliptin-Enalapril: Formulation, Optimization, and Comparative In Vitro-In Vivo Evaluation.

The current study aims at formulating and optimizing lipospheres (LS) by the Box-Behnken design (BBD) from safe biodegradable carnauba wax (CW) to co-administer saxagliptin (SG) and enalapril (EP) for co-existing chronic hypertensive diabetes in order to overcome inadequacies of conventional modes of drug administration. Optimized liposphere formulation (OLF) was selected by a numerical optimization procedure and a comparative in vivo pharmacokinetic study of OLF and commercial brands was also performed. Discrete, free-flowing, spherical, smooth-surface LS having a size range of 5-10 µm and zeta potential of - 20 to - 30 mV were successfully formulated. Compatibility studies by FTIR and DSC proved the lack of interaction of components while XRD suggested the transformation of crystalline drugs to amorphous form. Outcomes of dependent optimizing variables like percentage yield (30-90%), EP-release (32-92%), and SG-release (28-95%) followed a polynomial quadratic model. Pharmacokinetics studies indicated a significantly lower Cmax of EP (125.22 ± 6.32) and SG (75.63 ± 3.85) and higher mean Tmax values (9.4 h for EP and 10.73 h for SG) from OLF in comparison with reference brands of EP (257.54 ± 8.23 ng/mL) and SG (393.66 ± 2.97 ng/mL). Additionally, a potential rise in half-life and MRT of SG and EP was achieved reaching approximately 2- to 3-fold higher than noted for reference brands. Importantly, the enhanced Tmax and AUC0-24 specified the achievement of enhanced bioavailability of both drugs from LS. Consequently, such an innovative approach could not only control drug release in both in vitro and in vivo analyses but also maintain plasma drug concentration for a longer time without maximizing Cmax leading towards effective management of chronic illnesses.