PCSK9 inhibitors: a patent review 2018-2023.

INTRODUCTION: Proprotein convertase subtilisin/kexin 9 (PCSK9) plays a crucial role in breaking down the hepatic low-density lipoprotein receptor (LDLR), thereby influencing the levels of circulating low-density lipoprotein cholesterol (LDL-C). Consequently, inhibiting PCSK9 through suitable ligands has been established as a validated therapeutic strategy for combating hypercholesterolemia and cardiovascular diseases. AREA COVERED: Patent literature claiming novel compounds inhibiting PCSK9 disclosed from 2018 to June 2023 available in the espacenet database, which contains more than 150 million patent documents from over 100 patent-granting authorities worldwide. EXPERT OPINION: The undisputable beneficial influence of PCSK9 as a pharmacological target has prompted numerous private and public institutions to patent chemical frameworks as inhibitors of PCSK9. While several compounds have advanced to clinical trials for treating hypercholesterolemia, they have not completed these trials yet. These compounds must contend in a complex market where new, costly, and advanced drugs, such as monoclonal antibodies and siRNA, are prescribed instead of inexpensive and less potent statins.

Olive (Olea europaea L.) Seed as New Source of Cholesterol-Lowering Bioactive Peptides: Elucidation of Their Mechanism of Action in HepG2 Cells and Their Trans-Epithelial Transport in Differentiated Caco-2 Cells.

The production of olive oil has important economic repercussions in Mediterranean countries but also a considerable impact on the environment. This production generates enormous quantities of waste and by-products, which can be exploited as new raw materials to obtain innovative ingredients and therefore make the olive production more sustainable. In a previous study, we decided to foster olive seeds by generating two protein hydrolysates using food-grade enzymes, alcalase (AH) and papain (PH). These hydrolysates have shown, both in vitro and at the cellular level, antioxidant and antidiabetic activities, being able to inhibit the activity of the DPP-IV enzyme and modulate the secretion of GLP-1. Given the multifunctional behavior of peptides, both hydrolysates displayed dual hypocholesterolemic activity, inhibiting the activity of HMGCoAR and impairing the PPI of PCSK9/LDLR, with an IC50 equal to 0.61 mg/mL and 0.31 mg/mL for AH and PH, respectively. Furthermore, both samples restored LDLR protein levels on the membrane of human hepatic HepG2 cells, increasing the uptake of LDL from the extracellular environment. Since intestinal bioavailability is a key component of bioactive peptides, the second objective of this work is to evaluate the capacity of AH and PH peptides to be transported by differentiated human intestinal Caco-2 cells. The peptides transported by intestinal cells have been analyzed using mass spectrometry analysis, identifying a mixture of stable peptides that may represent new ingredients with multifunctional qualities for the development of nutraceuticals and functional foods to delay the onset of metabolic syndrome, promoting the principles of environmental sustainability.

Glycomacropeptide (GMP) rescued the oxidative and inflammatory activity of free L-AAs in human Caco-2 cells: New insights that support GMP as a valid and health-promoting product for the dietary management of phenylketonuria (PKU) patients.

Phenylketonuria represents the most prevalent inborn error of amino acid metabolism. In early diagnosed patients adequate and continued dietary treatment results in a good neurologic outcome. However, due to the natural protein and phenylalanine-restricted diet, oxidative stress represents a concern in phenylketonuric patients. Clear evidences suggest that the pathophysiology of PKU is also dependent by mitochondrial impairment and oxidative stress. In this context due to the tight connection between oxidative and inflammatory stress and noncommunicable diseases (NCDs) development, it is reasonable to hypothesize that PKU patients may present a higher risk to develop NCDs during their life. Currently available protein substitutes on the market include free amino acids (L-AAs), prolonged-release protein substitute and formula containing glycomacropeptide (GMP). Our results suggest that free L-AAs significanlty worsens the intestinal hydrogen peroxide (H2O2) and lipopolysaccharides (LPS)-induced oxidative and inflammatory status in Caco-2 cells, which are significantly restored towards physiological condition by GMP alone and when present in a 1:1 mixture with free L-AAs, providing new preclinical piece of information which can shed a shadow on the mechanism of action of these products on PKU patients and their future management.

Plant bioactive peptides for cardiovascular disease prevention.

Cardiovascular disease (CVD) is a major cause of deaths in industrialized countries and a constantly growing cause of morbidity and mortality worldwide Hypercholesterolemia is one of the main risk factors for CVD progression that may be prevented by lifestyle changes, including diet. This chapter will discuss the role of peptides from plants (soybean, lupin, cowpea, hempseed, and rice bran) sources with pleotropic activity for the prevention of CVD. Overall, the bioactivity that will be mainly discussed it is the hypocholesterolemic one. The very diversified structures of the hypocholesterolemic peptides so far identified explains the reason why they exert their activity through different mechanisms of action that will be extensively described in this review. Doubtlessly, their potential use in nutritional application is desirable, however, only few of them have been tested in vivo. Therefore, more efforts need to be pursued for singling out good candidates for the development of functional foods or dietary supplements.

Bioavailability Assessment of an Iron Formulation Using Differentiated Human Intestinal Caco-2 Cells.

In recent years, there has been growing interest in exploring alternative and innovative delivery systems to improve the efficacy of iron supplements, satisfying iron needs and lowering side effects. To address this issue, this study aimed at demonstrating the advantages of Ferro Supremo formulation (composed of encapsulated iron, vitamins, and micronutrients), in terms of capacity to improve iron intestinal absorption, in comparison with standard FeSO4. Hence, differentiated Caco-2 cells have been used for assessing the in vitro bioavailability and safety of FS and FeSO4. MTT experiments demonstrated that both FS and FeSO4 are not able to impair the viability of Caco-2 cells. Furthermore, the quantitative and qualitative analysis, conducted by atomic absorption spectrometry and fluorescence determinations, revealed that FS can enter, accumulate in the cytoplasm, and be transported by intestinal cells four times more efficiently than FeSO4. Our findings indicate that this formulation can be considered a valuable and efficiently good choice as food supplements for improving iron deficiency.

MOMAST® Reduces the Plasmatic Lipid Profile and Oxidative Stress and Regulates Cholesterol Metabolism in a Hypercholesterolemic Mouse Model: The Proof of Concept of a Sustainable and Innovative Antioxidant and Hypocholesterolemic Ingredient.

MOMAST® is a patented natural phenolic complex, rich in tyrosol (9.0 g/kg, Tyr), hydroxityrosol (43,5 g/kg, OH-Tyr), and verbascoside (5.0 g/Kg), which is obtained from the OVW by-product of the Coratina cultivar with potent direct antioxidant activity (measured by DPPH and FRAP assays, respectively). Indeed, MOMAST® represents an innovative sustainable bioactive ingredient which has been obtained with ethical and empowering behavior by applying the principles of a circular economy. In the framework of research aimed at fostering its health-promoting activity, in this study it was clearly demonstrated that MOMAST® treatment reduced the oxidative stress and levels of total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol, and increased the HDL levels, without changes in the triglyceride (TG) levels in Western diet (WD)-fed mice. The modulation of the plasmatic lipid profile is similar to red yeast rice (RYR) containing Monacolin K (3%). In addition, at the molecular level in liver homogenates, similarly to RYR, MOMAST® exerts cholesterol-lowering activity through the activation of LDL receptor, whereas, unlike RYR, MOMAST® reduces proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels via hepatic nuclear factor 1 (HNF1)-α activation. Hence, this study provides the proof of concept regarding the hypocholesterolemic activity of MOMAST, which could be successfully exploited as an active ingredient for the development of innovative and sustainable dietary supplements and functional foods.

Computational Mechanics of Form-Fitting 3D-Printed Lattice-Based Wrist-Hand Orthosis for Motor Neuron Disease.

Motor neuron disease (MND) patients often experience hand-wrist muscle atrophy resulting in severe social consequences and hampering their daily activities. Although hand-wrist orthosis is commonly used to assist weakened muscles, its effectiveness is limited due to the rapid progression of the disease and the need for customization to suit individual patient requirements. To address these challenges, this study investigates the application of three-dimensional (3D) printing technology to design and fabricate two lattice structures inspired by silkworm cocoons, using poly-ε-caprolactone as feedstock material. Finite element method (FEM) analysis is employed to study the mechanical behavior, enabling control over the geometric configuration incorporated into the hand-wrist orthosis. Through tensile displacement and three-point bending simulations, the stress distribution is examined for both lattice geometries. Geometry-1 demonstrates anisotropic behavior, while geometry-2 exhibits no strict directional dependence due to its symmetry and uniform node positioning. Moreover, the biocompatibility of lattices with human skin fibroblasts is investigated, confirming excellent biocompatibility. Lastly, the study involves semi-structured interviews with MND patients to gather feedback and develop prototypes of form-fitting 3D-printed lattice-based hand-wrist orthosis. By utilizing 3D printing technology, this study aims to provide customized orthosis that can effectively support weakened muscles and reposition the hand for individuals with MND.

Physicochemical and functional properties of rainbow trout (Oncorhynchus mykiss) hydrolysate.

Due to the continuous growth of the world population, there is an urgent need to find sustainable sources of high-quality protein. Fish side streams rich in essential nutrients and accounting for 60-70% of the whole fish, represent a sustainable source for recovery of valuable protein compounds. The present study aimed at extensive characterization of physicochemical, antioxidant and techno-functional properties of fish protein hydrolysate (FPH) obtained from farmed rainbow trout (Oncorhynchus mykiss). The FPH was produced from a minced rainbow trout raw material by enzymatic hydrolysis performed at 50 °C with addition of 0.05% w/w papain and 0.05% w/w bromelain. After inactivation of the proteases at 90 °C for 10 min, the content of the bioreactor was centrifuged, and the soluble protein fraction (FPH) was collected and freeze-dried. The total protein content of the FPH with 17.24% degree of hydrolysis was high (88.9%) and mainly represented by water-soluble proteins, while the lipid content was below 1%. In addition to the high protein content, trout hydrolysate had low protein oxidation values characterized by a relatively low total carbonyl content together with high amount of thiol groups (3.64 ± 0.31 and 20.7 ± 0.6 nmol/mg protein, respectively). No glass transition was detected in the differential scanning calorimetry (DSC) heat flow curves, suggesting lack of unfreezable solution formation in the FPH at freezing temperatures. The viscosity of FPH showed typical Newtonian behaviour. A peptidomic investigation (using HPLC-MS/MS technique) displayed chemical composition of the trout hydrolysate and identified peptide sequences which are present in the hydrolysate mixture, as well as proteins to which each peptide belongs to. In conclusion, it was suggested to use the obtained trout hydrolysate as a functional ingredient in the food and nutraceutical industry.

Evaluation of the multifunctional dipeptidyl-peptidase IV and angiotensin converting enzyme inhibitory properties of a casein hydrolysate using cell-free and cell-based assays.

The objective of the study was the evaluation of the potential pleiotropic effect of a commercial casein hydrolysate (CH). After an analysis of the composition, the BIOPEP-UWM database suggested that these peptides contained numerous sequences with potential inhibitory activities on angiotensin converting enzyme (ACE) and dipeptidyl-peptidase IV (DPP-IV). The anti-diabetic and anti-hypertensive effects of these peptides were thus assessed using either cell-free or cell-based assays. In the cell-free system, CH displayed inhibitory properties against DPP-IV (IC50 value equal to 0.38 ± 0.01 mg/mL) and ACE (IC50 value equal to 0.39 ± 0.01 mg/mL). Further, CH reduced the DPP-IV and ACE activities expressed by human intestinal Caco-2 cells by 61.10 ± 1.70% and 76.90 ± 4.47%, respectively, versus untreated cells, after 6 h of treatment at the concentration of 5 mg/mL. This first demonstration of the multifunctional behavior of this material suggests that it may become an anti-diabetic and/or anti-hypertensive ingredient to be included in the formulation of different functional food or nutraceutics.

Chemical and biological characterization of the DPP-IV inhibitory activity exerted by lupin (Lupinus angustifolius) peptides: From the bench to the bedside investigation.

Dipeptidyl peptidase IV (DPP-IV) is considered a key target for the diabetes treatment, since it is involved in glucose metabolism. Although lupin protein consumption shown hypoglycemic activity, there is no evidence of its effect on DPP-IV activity. This study demonstrates that a lupin protein hydrolysate (LPH), obtained by hydrolysis with Alcalase, exerts anti-diabetic activity by modulating DPP-IV activity. In fact, LPH decreased DPP-IV activity in a cell-free and cell-based system. Contextually, Caco-2 cells were employed to identify LPH peptides that can be intestinally trans-epithelial transported. Notably, 141 different intestinally transported LPH sequences were identified using nano- and ultra-chromatography coupled to mass spectrometry. Hence, it was demonstrated that LPH modulated the glycemic response and the glucose concentration in mice, by inhibiting the DPP-IV. Finally, a beverage containing 1 g of LPH decreased DPP-IV activity and glucose levels in humans.

Computational Design, Synthesis, and Biological Evaluation of Diimidazole Analogues Endowed with Dual PCSK9/HMG-CoAR-Inhibiting Activity.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is responsible for the degradation of the hepatic low-density lipoprotein receptor (LDLR), which regulates circulating cholesterol levels. Consequently, the PCSK9 inhibition is a valuable therapeutic approach for the treatment of hypercholesterolemia and cardiovascular diseases. In our studies, we discovered Rim13, a polyimidazole derivative reducing the protein-protein interaction between PCSK9 and LDLR with an IC50 of 1.6 µM. The computational design led to the optimization of the shape of the PCSK9/ligand complementarity, enabling the discovery of potent diimidazole derivatives. In fact, carrying out biological assays to fully characterize the cholesterol-lowering activity of the new analogues and using both biochemical and cellular techniques, compound Dim16 displayed improved PCSK9 inhibitory activity (IC50 0.9 nM). Interestingly, similar to other lupin-derived peptides and their synthetic analogues, some compounds in this series showed dual hypocholesterolemic activity since some of them complementarily inhibited the 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Antioxidant Effect Assessment and Trans Epithelial Analysis of New Hempseed Protein Hydrolysates.

Hempseed (Cannabis sativa) is one of the most promising sources of plant proteins. It contains approximately 24% (w/w) protein, and edestin accounts for approximately 60-80% (w/w) of its total proteins. In a framework of research aimed at fostering the proteins recovered from the press cake by-products generated after the extraction of hempseed oil, two hempseed protein hydrolysates (HH1 and HH2) were produced at an industrial level using a mixture of different enzymes from Aspergillus niger, Aspergillus oryzae, and Bacillus licheniformis for different times (5 h and 18 h). Using a combination of different direct antioxidant tests (DPPH, TEAC, FRAP, and ORAC assays, respectively), it has been demonstrated that HHs exert potent, direct antioxidant activity. A crucial feature of bioactive peptides is their intestinal bioavailability; for this reason, in order to solve this peculiar issue, the ability of HH peptides to be transported by differentiated human intestinal Caco-2 cells has been evaluated. Notably, by using mass spectrometry analysis (HPLC Chip ESI-MS/MS), the stable peptides transported by intestinal cells have been identified, and dedicated experiments confirmed that the trans-epithelial transported HH peptide mixtures retain their antioxidant activity, suggesting that these hempseed hydrolysates may be considered sustainable antioxidant ingredients to be exploited for further application, i.e., nutraceutical and/or food industries.

Virgin Olive Oil By-Product Valorization: An Insight into the Phenolic Composition of Olive Seed Extracts from Three Cultivars as Sources of Bioactive Molecules.

Olives are very rich in phenolic compounds with important health-promoting properties. The profile and content of phenols in olive pulp and virgin olive oil are strongly influenced by the fruit ripening degree, but little is known concerning the evolution of phenolic compounds in the seed. In this work, the phenolic composition of seed from Tuscan cultivars (Frantoio, Moraiolo, Leccino) was studied over maturation. Starting from each seed sample, a phenolic extract was prepared and analyzed by HPLC-DAD-MS. Nüzhenide and nüzhenide 11-methyl oleoside were by far the most abundant phenolic compounds; their content reached up to 46 g/kg in dry seeds, although this diminished in the final stage of fruit maturation. At the same time, the phenolic composition of the pulp was also characterized over the course of maturation, showing that oleuropein was by far the most abundant compound, with concentrations comparable to those of nüzhenide and nüzhenide 11-methyl oleoside in the seeds. Overall, the total amount of phenols in seed dry extracts was significant, reaching approx. 100 g/kg. The chemically characterized dry phenolic extracts from seeds could be used for future biological assays aimed at evaluating the potential bioactivities of these phytocomplexes.

Rainbow Trout (Oncorhynchus mykiss) as Source of Multifunctional Peptides with Antioxidant, ACE and DPP-IV Inhibitory Activities.

The present study aimed at characterizing the possible biological activities of the multifunctional low molecular weight fractions (<3 kDa) peptides isolated from rainbow trout (Oncorhynchus mykiss) obtained by enzymatic hydrolysis. The fish protein hydrolysate (FPH) was tested for its antioxidant property along with its angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities. In particular, the 2,2-diphenyl-1-picrylhydrazyl (DPPH), the ferric reducing antioxidant power (FRAP), the oxygen radical absorbance capacity (ORAC) assay and the 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays were carried out for the evaluation of the in vitro antioxidant activity. The cell-free ACE and DPP-IV inhibitory activity assays were also estimated, showing a dose-dependent inhibition. These biological properties were additionally quantified at the cellular level using human intestinal Caco-2 cells. Namely, the antioxidant activity was determined by evaluating the capability of the hydrolysate to reduce the H2O2-induced reactive oxygen species (ROS) and lipid peroxidation levels, and the DPP-IV activity assays show a reduction of enzyme activity of up to 27.57 ± 3.7% at 5 mg/mL. The results indicate that Oncorhynchus mykiss-derived peptides may have potential employment as health-promoting ingredients.

Valorization of the Antioxidant Effect of Mantua PGI Pear By-Product Extracts: Preparation, Analysis and Biological Investigation.

For improving the management of the production chain of PGI Mantua pears (which comprises many varieties, including Abate Fetel), applying the cardinal principles of circular economy and sustainability, the fruits with diseases or defects were recovered for producing dried rounds of pears from the Abate Fetel cultivar, a new product with high nutritional value that extends the remaining life. This process led to the production of secondary and residual by-products, which are mainly composed of the highest and lowest part of the fruits, comprising seeds, pulps, peels and petioles. Hence, this study was focused on the valorization of these secondary by-products of Abate Fetel pears through the production of pear extracts using traditional and "green" extraction methods that involve the use of supercritical CO2 fluid extraction. The produced extracts, together with a reference solvent-derived extract, were analyzed by HPLC-ESI-MS, and in parallel, their direct and cellular antioxidant activity were assessed. Evidence has indicated that all the tested extracts reduced the H2O2-induced reactive oxygen species (ROS), lipid peroxidation and nitric oxide (NO) levels, respectively, in human intestinal Caco-2 cells. Hence, this study clearly suggests that extracts obtained from Mantuan PGI pear by-products may be used as valuable sources of bioactive upcycled phytocomplex for the development of dietary supplements and/or functional foods.

Isolation and functional characterization of hemp seed protein-derived short- and medium-chain peptide mixtures with multifunctional properties for metabolic syndrome prevention.

This study aims to obtain a valuable mixture of short-chain peptides from hempseed as a new ingredient for developing nutraceutical and functional foods useful for preventing metabolic syndrome that represents the major cause of death globally. A dedicated analytical platform based on a purification step by size exclusion chromatography or ultrafiltration membrane and high-resolution mass spectrometry was developed to isolate and comprehensively characterize short-chain peptides leading to the identification of more than 500 short-chain peptides. Our results indicated that the short-chain peptide mixture was about three times more active than the medium-chain peptide mixture and total hydrolysate with respect to measured inhibition of the angiotensin-converting enzyme. The short-chain peptide mixture was also two times more active as a dipeptidyl peptidase IV, and twofold more active on the cholesterol metabolism pathway through the modulation of low-density lipoprotein receptor.

Quality Assessment of the Protein Ingredients Recovered by Ultrasound-Assisted Extraction from the Press Cakes of Coconut and Almond Beverage Preparation.

The manufacture of vegetal beverages has the drawback of producing large amounts of press cakes that are generally used as feed components. This work had the objective of valorizing the press cakes deriving from almond and coconut drinks production by using ultrasound-assisted extraction (UAE) to obtain protein ingredients for human use. Starting from coconut and almond press cakes, whose initial protein contents were 19.7% and 18.6%, respectively, the UAE treatment allowed liquid fractions to be obtained that were then freeze-dried: the extraction yields were 24.4 g dry extract/100 g press cake in case of coconut and 49.3 g dry extract/100 g press cake in case of almond. The protein contents of these dried materials were 30.10% and 22.88%, respectively. The quality of the extracted protein ingredients was assessed in term of phytic acid content, protein profile, techno-functional features, and antioxidant properties. The sonication had also a favorable effect on digestibility.

Improving Stability Enhances In Vivo Efficacy of a PCSK9 Inhibitory Peptide.

Optimization of peptide stability is essential for the development of peptides as bona fide alternatives to approved monoclonal antibodies. This is clearly the case for the many peptides reported to antagonize proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a clinically validated target for lowering cholesterol. However, the effects of optimization of stability on in vivo activity and particularly the effects of binding to albumin, an emerging drug design paradigm, have not been studied for such peptide leads. In this study, we optimized a PCSK9 inhibitory peptide by mutagenesis and then by conjugation to a short lipidated tag to design P9-alb fusion peptides that have strong affinity to human serum albumin. Although attachment of the tag reduced activity against PCSK9, which was more evident in surface plasmon resonance binding and enzyme-linked immunosorbent competition assays than in cellular assays of activity, activity remained in the nanomolar range (∼40 nM). P9-alb peptides were exceptionally stable in human serum and had half-lives exceeding 48 h, correlating with longer half-lives in mice (40.8 min) compared to the unconjugated peptide. Furthermore, the decrease in in vitro binding was not deleterious to in vivo function, showing that engendering albumin binding improved low-density lipoprotein receptor recovery and cholesterol-lowering activity. Indeed, the peptide P9-albN2 achieved similar functional endpoints as the approved anti-PCSK9 antibody evolocumab, albeit at higher doses. Our study illustrates that optimization of stability instead of binding affinity is an effective way to improve in vivo function.

Discovery of a Potent and Highly Selective Dipeptidyl Peptidase IV and Carbonic Anhydrase Inhibitor as "Antidiabesity" Agents Based on Repurposing and Morphing of WB-4101.

The management of patients with type 2 diabetes mellitus (T2DM) is shifting from cardio-centric to weight-centric or, even better, adipose-centric treatments. Considering the downsides of multidrug therapies and the relevance of dipeptidyl peptidase IV (DPP IV) and carbonic anhydrases (CAs II and V) in T2DM and in the weight loss, we report a new class of multitarget ligands targeting the mentioned enzymes. We started from the known α1-AR inhibitor WB-4101, which was progressively modified through a tailored morphing strategy to optimize the potency of DPP IV and CAs while losing the adrenergic activity. The obtained compound 12 shows a satisfactory DPP IV inhibition with a good selectivity CA profile (DPP IV IC50: 0.0490 µM; CA II Ki 0.2615 µM; CA VA Ki 0.0941 µM; CA VB Ki 0.0428 µM). Furthermore, its DPP IV inhibitory activity in Caco-2 and its acceptable pre-ADME/Tox profile indicate it as a lead compound in this novel class of multitarget ligands.