When Kinases Meet PROTACs

Small molecule drugs targeting kinases have revolutionized treatment options for millions of patients worldwide, especially in oncology. These targeted treatments have less side effects because they inhibit a specific dysfunctional kinase usually with relatively narrow selectivity. However, kinase inhibitors do have well‐established liabilities, most prominently the emergence of drug resistance. Moreover, the majority of kinases are multidomain and multifunctional proteins that in addition to their enzymatic activity have scaffolding and other roles, and inhibitors seldom address these alternative functions. Recently, small molecule mediated targeted protein degradation emerged as a new pharmacological strategy. The majority of small molecule degraders are bispecific molecules called proteolysis targeting chimeras (PROTACs), and their mechanism of action is based on simultaneous recruitment of the target of interest and an E3 ligase, resulting in target polyubiquitination and eventual destruction by the proteasome. Over the last couple of years, PROTAC strategy has been developed and validated for a range of targets, including kinases. Here, we introduce the targeted protein degradation strategy, provide an overview of representative kinase PROTACs, and describe design rationales, efficacy and specificity. We also discuss their potential advantages, as well as comment on some of the limitations of this emerging pharmacological modality.     

Comparative Evaluation of Intestinal Absorption and Functional Value of Iron Dietary Supplements and Drug with Different Delivery Systems

Iron is a fundament micronutrient, whose homeostasis is strictly regulated. Iron deficiency anemia is among the most widespread nutritional deficiencies and its therapy, based on dietary supplement and drugs, may lead to severe side effects. With the aim of improving iron bioavailability while reducing iron oral therapy side effects, novel dietary supplements based on innovative technologies—microencapsulation, liposomes, sucrosomes—have been produced and marketed. In the present work, six iron dietary supplements for different therapeutic targets were compared in terms of bioaccessibility, bioavailability, and safety by using an integrated in vitro approach. For general-purpose iron supplements, ME + VitC (microencapsulated) showed a fast, burst intestinal iron absorption kinetic, which maintained iron bioavailability and ferritin expression constant over time. SS + VitC (sucrosomes), on the other side, showed a slower, time-dependent iron absorption and ferritin expression trend. ME + Folate (microencapsulated) showed a behavior similar to that of ME + VitC, albeit with a lower bioavailability. Among pediatric iron supplements, a time-dependent bioavailability increase was observed for LS (liposome), while PIC (polydextrose-iron complex) bioavailability is severely limited by its poor bioaccessibility. Finally, except for SS + VitC, no adverse effects on intestinal mucosa vitality and barrier integrity were observed. Considering obtained results and the different therapeutic targets, microencapsulation-based formulations are endowed with better performance compared to the other formulations. Furthermore, performances of microencapsulated products were obtained with a lower iron daily dose, limiting the potential onset of side effects.     

Synthetic approaches to constructing proteolysis targeting chimeras (PROTACs)

The development of various heterobifunctional constructs dubbed PRoteolysis-TArgeting Chimeras (PROTACs) has gained a significant impetus in the last few years. A viable alternative to the traditional occupancy-based inhibition of aberrantly hyperactive proteins, PROTACs operate by an event-based catalytic mechanism bringing together the protein of interest (POI, to be degraded) and E3 ubiquitin ligases. The formation of the ternary complex ‘POI–PROTAC–E3 ubiquitin ligase’ is the critical step which leads to the ubiquitination of the POI and its proteasomal degradation. The current Focused Review aims to highlight the syntheses of selected innovative PROTAC-type degraders of the therapeutically important protein targets as well as some notable chemical aspects of PROTAC construction. The overview is focusing on PROTACs aimed at recruiting Cereblon, the most exploited E3 ligase for targeted protein degradation.     

Improving on Nature: Making a Cyclic Heptapeptide Orally Bioavailable

The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR‐derived structures, amide H–D exchange rates, and temperature‐dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N‐methylation.     

Development of an LC-MS/MS Method for ARV-110, a PROTAC Molecule,and Applications to Pharmacokinetic Studies

ARV-110, a novel proteolysis-targeting chimera (PROTAC), has been reported to show satisfactory safety and tolerability for prostate cancer therapy in phase I clinical trials. However, there is a lack of bioanalytical assays for ARV-110 determination in biological samples. In this study, we developed and validated an LC-MS/MS method for the quantitation of ARV-110 in rat and mouse plasma and applied it to pharmacokinetic studies. ARV-110 and pomalidomide (internal standard) were extracted from the plasma samples using the protein precipitation method. Sample separation was performed using a C18 column and a mobile phase of 0.1% formic acid in distilled water–0.1% formic acid in acetonitrile (30:70, v/v). Multiple reaction monitoring was used to quantify ARV-110 and pomalidomide with ion transitions at m/z 813.4 → 452.2 and 273.8 → 201.0, respectively. The developed method showed good linearity in the concentration range of 2–3000 ng/mL with acceptable accuracy, precision, matrix effect, process efficiency, and recovery. ARV-110 was stable in rat and mouse plasma under long-term storage, three freeze-thaw cycles, and in an autosampler, but unstable at room temperature and 37 °C. Furthermore, the elimination of ARV-110 via phase 1 metabolism in rat, mouse, and human hepatic microsomes was shown to be unlikely. Application of the developed method to pharmacokinetic studies revealed that the oral bioavailability of ARV-110 in rats and mice was moderate (23.83% and 37.89%, respectively). These pharmacokinetic findings are beneficial for future preclinical and clinical studies of ARV-110 and/or other PROTACs.     

Preparation,Characterization, and In Vivo Evaluation of Amorphous Icaritin Nanoparticles Prepared by a Reactive Precipitation Technique

Icaritin is a promising anti-hepatoma drug that is currently being tested in a phase-III clinical trial. A novel combination of amorphization and nanonization was used to enhance the oral bioavailability of icaritin. Amorphous icaritin nanoparticles (AINs) were prepared by a reactive precipitation technique (RPT). Fourier transform infrared spectrometry was used to investigate the mechanism underlying the formation of amorphous nanoparticles. AINs were characterized via scanning electron microscopy, X-ray powder diffraction, and differential scanning calorimetry. Our prepared AINs were also evaluated for their dissolution rates in vitro and oral bioavailability. The resultant nanosized AINs (64 nm) were amorphous and exhibited a higher dissolution rate than that derived from a previous oil-suspension formulation. Fourier transform infrared spectroscopy (FTIR) revealed that the C=O groups from the hydrophilic chain of polymers and the OH groups from icaritin formed hydrogen bonds that inhibited AIN crystallization and aggregation. Furthermore, an oral administration assay in beagle dogs showed that C_max and AUC_last of the dried AINs formulation were 3.3-fold and 4.5-fold higher than those of the oil-suspension preparation (p < 0.01), respectively. Our results demonstrate that the preparation of amorphous drug nanoparticles via our RPT may be a promising technique for improving the oral bioavailability of poorly water-soluble drugs.     

An innovative rhein-matrine cocrystal: Synthesis,characterization, formation mechanism and pharmacokinetic study

Rhein (Rhe), an anthraquinone derivative, exhibits excellent anti-inflammatory effects and other pharmacological activities, but its clinical application remains limited due to poor solubility. The present work aims at the improvement of solubility and oral bioavailability of Rhe through cocrystal formation. For this purpose, Rhe and matrine (Mat) were selected as pharmaceutical ingredient (API) and cocrystal former (CCF), respectively, and the Rhe-Mat cocrystal was synthesized and characterized by single crystal X-ray diffraction (SXRD), powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC). The formation mechanism of Rhe-Mat cocrystal was elucidated by molecular surface electrostatic potential (MSEP). It is worth mentioning that the 50-fold increment of dissolution in vitro was observed in pure water in the form of Rhe-Mat cocrystal. Furthermore, the in vivo studies revealed that Rhe-Mat cocrystal indicated the faster absorption rate and the higher peak blood concentration than the pure Rhe. Hence, it can be concluded that current study successfully improved the solubility and oral bioavailability of Rhe.     

Targeted Degradation of PD-L1 and Activation of the STING Pathway by Carbon-Dot-Based PROTACs for Cancer Immunotherapy

Proteolysis targeting chimeras (PROTACs) technology is an emerging approach to degrade disease-associated proteins. Here, we report carbon-dot (CD)-based PROTACs (CDTACs) that degrade membrane proteins via the ubiquitin-proteasome system. CDTACs can bind to programmed cell death ligand 1 (PD-L1), recruit cereblon (CRBN) to induce PD-L1 ubiquitination, and degrade them with proteasomes. Fasting-mimicking diet (FMD) is also used to enhance the cellular uptake and proteasome activity. More than 99 % or 90 % of PD-L1 in CT26 or B16-F10 tumor cells can be degraded by CDTACs, respectively. Furthermore, CDTACs can activate the stimulator of interferon genes (STING) pathway to trigger immune responses. Thus, CDTACs with FMD treatment effectively inhibit the growth of CT26 and B16-F10 tumors. Compared with small-molecule-based PROTACs, CDTACs offer several advantages, such as efficient membrane protein degradation, targeted tumor accumulation, immune system activation, and in vivo detection.     

Determination of Trace Metoclopramide by Anodic Stripping Voltammetry with Nafion Modified Glassy Carbon Electrode

Metoclopramide[monohydrate of 4-amino-5-chloro-N-(2-diethylaminoethyl)-2-methoxybenzamide hydrochloride,MCP] is the active ingredient of many pharmaceutical preparations concermed with the modification of digestive behaviour. Studies describing the pharmacokinetics and disposition of MCP in humans indicated that the drug is rapidly and well absorbed following oral administration with peak time at about one hour. However,the bioavailability of MCP is very variable (32-97%) and this out of considerable inter-individual variation in metabolism. The difference in the bioavailability of oral MCP in different subjects has potential clinical importance since its antiemetic effect and central nervous system adverse effects correlate with the plasma concentrations. Therefore,the measurement of MCP plasma concentration is essential to optimize therapy and to avoid toxic concentrations.     

Biological regulation on synovial fibroblast and the treatment of rheumatoid arthritis by nobiletin-loaded tetrahedral framework nucleic acids cargo tank

The hyperplasia and destruction of synovial tissue have an important impact on the development of rheumatoid arthritis (RA), the abnormal proliferation and migration of synovial fibroblast in synovial tissue is similar to tumor cells. Targeting anomalous synovial fibroblast and designing a high bioavailability nano drug delivery system can reduce the dosage for the treatment of rheumatoid arthritis and it is of great significance to reduce toxic and side effects and improve curative effect. In this experiment, the nobiletin-loaded tetrahedral framework nucleic acids cargo tank was established, carrying anti-inflammatory small molecule monomer drug nobiletin with minimal bioavailability. Both in vitro cell experiments and in vivo animal studies proved the nano cargo tank enhance the role of nobiletin in reducing the invasiveness of pathological synovial fibroblast and promote their apoptosis, effectively alleviate the disease development of rheumatoid arthritis.     

The complexation of insulin with sodium N‐[8‐(2‐hydroxybenzoyl)amino]‐caprylate for enhanced oral delivery: Effects of concentration,ratio, and pH

Permeation enhancers (PEs), such as N-[8-(2-hydroxybenzoyl)amino]-caprylate (SNAC), have been reported to improve the oral absorption of various macromolecules. However, the bioavailabilities of these formulations are quite low and variable due to the influences of enzymes, pH and other gastrointestinal barriers. In this study, we revealed that SNAC could interact with insulin to form tight complexes in a specific concentration (insulin ≥ 40 µg/mL)-, ratio (SNAC/insulin ≥ 20:1)- and pH (≥ 6.8)-dependent manner, thus contributing to a significantly high efficacy of oral insulin delivery. Specifically, absorption mechanism studies revealed that the SNAC/insulin complexes were internalized into the cells by passive diffusion and remained intact when transported in the cytosol. Furthermore, the complexes accelerated the exocytosis of insulin to the basolateral side, thereby enhancing its intestinal mucosal permeability. Eudragit^? S100-entrapped SNAC/insulin microspheres were then prepared and exhibited an apparent permeability coefficient (P_app) that was 6.6-fold higher than that of the insulin solution. In diabetic rats, hypoglycemic activity was sustained for more than 10 h after the microspheres were loaded into enteric-coated capsules. Further pharmacokinetic studies revealed an approximately 6.3% oral bioavailability in both the fasted and fed states, indicating a negligible food effect. Collectively, this study provides insight into the interaction between PEs and payloads and presents an SNAC-based oral insulin delivery system that has high oral bioavailability and patient-friendly medication guidance.     

A critical review of the bioavailability of glucosinolates and related compounds

Glucosinolates (GLSs) are relatively inert (Z)-N-hydroximinosulfate esters, possessing a sulfur-linked beta-D-glucopyranose moiety and a variable side chain, found almost exclusively in cruciferous vegetables. Following cell disruption, they are hydrolysed by plant myrosinases, forming a group of chemically reactive and biologically active compounds. There is considerable evidence that these breakdown products, when consumed in the diet, may affect the risk of developing chronic diseases. However, in order for any compound to exert an activity in vivo, it is necessary to reach the site of action in an appropriate form and sufficient concentration. Deleterious and toxic effects may be observed at high concentrations: hence, bioavailability is a key factor defining the physiological, beneficial dose window of GLS hydrolysis products (GLS-HPs). For some GLS-HPs, this window can be rather narrow, and therefore is a critical parameter to be considered. In this review we critically evaluate the present state of knowledge on all factors that affect bioavailability of GLS-HPs. This includes liberation from the plant material, absorption from the digestive system, distribution around the body, metabolism and excretion.     

Design,Synthesis, and Biological Evaluation of HDAC Degraders with CRBN E3 Ligase Ligands

Histone deacetylases (HDACs) play important roles in cell growth, cell differentiation, cell apoptosis, and many other cellular processes. The inhibition of different classes of HDACs has been shown to be closely related to the therapy of cancers and other diseases. In this study, a series of novel CRBN-recruiting HDAC PROTACs were designed and synthesized by linking hydroxamic acid and benzamide with lenalidomide, pomalidomide, and CC-220 through linkers of different lengths and types. One of these PROTACs, denoted 21a, with a new benzyl alcohol linker, exhibited comparably excellent HDAC inhibition activity on different HDAC classes, acceptable degradative activity, and even better in vitro anti-proliferative activities on the MM.1S cell line compared with SAHA. Moreover, we report for the first time the benzyl alcohol linker, which could also offer the potential to be used to develop more types of potent PROTACs for targeting more proteins of interest (POI).     

喜树碱及其衍生物的聚乙二醇化学修饰药物研究进展

目的：喜树碱及其衍生物药物是一类重要的抗肿瘤药物,但其存在水溶性差、生物利用度低、毒副作用大等问题,喜树碱及其衍生物的聚乙二醇化学修饰具有十分诱人的前景,但目前国内研究较少,本文综述近年来喜树碱及其衍生物药物的聚乙二醇化学修饰研究进展。方法：以喜树碱（Camptothecin, CPT）、伊立替康（Irinotecan, CPT-11）、7-乙基-10-羟基喜树碱（7-ethyl-10-hydroxycamptothecin, SN38）、聚乙二醇（Polyethylene glycol, PEG）、聚乙二醇化喜树碱（PEG-CPT）等为关键词,查阅近几年国内外相关研究文献。结果与结论：综述了喜树碱及其衍生物的聚乙二醇化学修饰药物的研究进展与临床研究报道,为发掘结构明确、组成稳定的新型化学修饰药物提供理论基础和技术支撑。     

Improved Safety and Anti-Glioblastoma Efficacy of CAT3-Encapsulated SMEDDS through Metabolism Modification

13a-(S)-3-pivaloyloxyl-6,7-dimethoxyphenanthro(9,10-b)-indolizidine (CAT3) is a novel oral anti-glioma pro-drug with a potent anti-tumor effect against temozolomide-resistant glioma. 13a(S)-3-hydroxyl-6,7-dimethoxyphenanthro(9,10-b)-indolizidine (PF403) is the active in vivo lipase degradation metabolite of CAT3. Both CAT3 and PF403 can penetrate the blood–brain barrier to cause an anti-glioma effect. However, PF403, which is produced in the gastrointestinal tract and plasma, causes significant gastrointestinal side effects, limiting the clinical application of CAT3. The objective of this paper was to propose a metabolism modification for CAT3 using a self-microemulsifying drug delivery system (SMEDDS), in order to reduce the generation of PF403 in the gastrointestinal tract and plasma, as well as increase the bioavailability of CAT3 in vivo and the amount of anti-tumor substances in the brain. Thus, a CAT3-loaded self-microemulsifying drug delivery system (CAT3-SMEDDS) was prepared, and its physicochemical characterization was systematically carried out. Next, the pharmacokinetic parameters of CAT3 and its metabolite in the rats’ plasma and brain were measured. Furthermore, the in vivo anti-glioma effects and safety of CAT3-SMEDDS were evaluated. Finally, Caco-2 cell uptake, MDCK monolayer cellular transfer, and the intestinal lymphatic transport mechanisms of SMEDDS were investigated in vitro and in vivo. Results show that CAT3-SMEDDS was able to form nanoemulsion droplets in artificial gastrointestinal fluid within 1 min, displaying an ideal particle size (15–30 nm), positive charge (5–9 mV), and controlled release behavior. CAT3-SMEDDS increased the membrane permeability of CAT3 by 3.9-fold and promoted intestinal lymphatic transport. Hence, the bioavailability of CAT3 was increased 79% and the level of its metabolite, PF403, was decreased to 49%. Moreover, the concentrations of CAT3 and PF403 were increased 2–6-fold and 1.3–7.2-fold, respectively, in the brain. Therefore, the anti-glioma effect in the orthotopic models was improved with CAT3-SMEDDS compared with CAT3 in 21 days. Additionally, CAT3-SMEDDS reduced the gastrointestinal side effects of CAT3, such as severe diarrhea, necrosis, and edema, and observed less inflammatory cell infiltration in the gastrointestinal tract, compared with the bare CAT3. Our work reveals that, through the metabolism modification effect, SMEDDS can improve the bioavailability of CAT3 and reduce the generation of PF403 in the gastrointestinal tract and plasma. Therefore, it has the potential to increase the anti-glioma effect and reduce the gastrointestinal side effects of CAT3 simultaneously.     

Small‐Molecule PROTACS: New Approaches to Protein Degradation

The current inhibitor‐based approach to therapeutics has inherent limitations owing to its occupancy‐based model: 1) there is a need to maintain high systemic exposure to ensure sufficient in vivo inhibition, 2) high in vivo concentrations bring potential for off‐target side effects, and 3) there is a need to bind to an active site, thus limiting the drug target space. As an alternative, induced protein degradation lacks these limitations. Based on an event‐driven model, this approach offers a novel catalytic mechanism to irreversibly inhibit protein function by targeting protein destruction through recruitment to the cellular quality control machinery. Prior protein degrading strategies have lacked therapeutic potential. However, recent reports of small‐molecule‐based proteolysis‐targeting chimeras (PROTACs) have demonstrated that this technology can effectively decrease the cellular levels of several protein classes.     

Oral Bioavailability of Flutamide from 1‐O‐Alkylglycerol Stabilized o/w Nanoemulsions

Size of oil globule and emulsifier film covering it are the key factors modifying oral absorption of drugs dissolved/dispersed in oil globules of o/w emulsions. Since oral bioavailability of flutamide (FTM) is poor, nanoemulsions incorporating FTM in oil phase and globules stabilized by a novel lipophilic emulsifier, 1‐O‐alkylglycerol were developed and characterized. All nanoemulsions had a size around 200 nm. They exhibited considerable release retardation in vitro. Flutamide nanoemulsions were evaluated for oral bioavailability in rabbits and in situ intestinal absorption in rats. Serum level data of flutamide obtained in the in situ experiments indicate higher FTM absorption from nanoemulsions than the aqueous FTM suspension. Among the nanoemulsions, 1‐O‐alkylglycerol stabilized systems showed higher absorption. The results of bioavailability studies in rabbits indicate increased absorption and decreased elimination of FTM from nanoemulsions, thus improving the bioavailability. Increased rate of absorption of flutamide in the in situ experiments can be attributed to the permeation enhancement effect of 1‐O‐alkylglycerols. However, size of the oil droplet seems to be the major factor in improving oral bioavailability in rabbits.     

Heavy Metals and Human Health: Possible Exposure Pathways and the Competition for Protein Binding Sites

Heavy metals enter the human body through the gastrointestinal tract, skin, or via inhalation. Toxic metals have proven to be a major threat to human health, mostly because of their ability to cause membrane and DNA damage, and to perturb protein function and enzyme activity. These metals disturb native proteins’ functions by binding to free thiols or other functional groups, catalyzing the oxidation of amino acid side chains, perturbing protein folding, and/or displacing essential metal ions in enzymes. The review shows the physiological and biochemical effects of selected toxic metals interactions with proteins and enzymes. As environmental contamination by heavy metals is one of the most significant global problems, some detoxification strategies are also mentioned.     

Stimuli-Responsive PROTACs for Controlled Protein Degradation

Proteolysis Targeting Chimeras (PROTACs) represent a promising therapeutic modality to address undruggable and resistant issues in drug discovery. However, potential on-target toxicity remains clinically challenging. We developed a generalized caging strategy to synthesize a series of stimuli-responsive PROTACs (sr-PROTACs) with diverse molecular blocks bearing robust and cleavable linkers, presenting “turn on” features in manipulating protein degradation. By leveraging pathological cues, such as elevated ROS, phosphatase, H₂S, or hypoxia, and external triggers, such as ultraviolet light, X-Ray, or bioorthogonal reagents, we achieved site-specific activation and traceless release of original PROTACs through de-caging and subsequent self-immolative cleavage, realizing selective uptake and controlled protein degradation in vitro. An in vivo study revealed that two sr-PROTACs with phosphate- and fluorine-containing cages exhibited high solubility and long plasma exposure, which were specifically activated by tumor overexpressing phosphatase or low dosage of X-Ray irradiation in situ, leading to efficient protein degradation and potent tumor remission. With more reactive biomarkers to be screened from clinical practice, our caging library could provide a general tool to design activatable PROTACs, prodrugs, antibody-drug conjugates, and smart biomaterials for personalized treatment, tissue engineering or regenerative medicine.