Four model polymers, representing (i) amorphous homopolymers , (ii) amorphous heteropolymers , (iii) semi-crystalline homopolymers , and (iv) semi-crystalline heteropolymers , were examined for their effectiveness in creating posaconazole-based amorphous solid dispersions (ASDs). Posaconazole (POS) is a triazole antifungal drug that has activity against Candida and Aspergillus species, belonging to class II of the biopharmaceutics classification system (BCS). This means that this active pharmaceutical ingredient (API) is characterized by solubility-limited bioavailability. Thus, one of the aims of its formulation as an ASD was to improve its aqueous solubility. Investigations were performed into how polymers affected the following characteristics: melting point depression of the API, miscibility and homogeneity with POS, improvement of the amorphous API’s physical stability, melt viscosity , extrudability, API content in the extrudate, long term physical stability of the amorphous POS in the binary drug–polymer system (in the form of the extrudate), solubility, and dissolution rate of hot melt extrusion (HME) systems. The obtained results led us to conclude that the physical stability of the POS-based system increases with the increasing amorphousness of the employed excipient. Copolymers, compared to homopolymers, display greater homogeneity of the investigated composition. However, the enhancement in aqueous solubility was significantly higher after utilizing the homopolymeric, compared to the copolymeric, excipients. Considering all of the investigated parameters, the most effective additive in the formation of a POS-based ASD is an amorphous homopolymer—K30. Improving the aqueous solubility and dissolution rate of poorly water-soluble active pharmaceutical ingredients (APIs), and new chemical entities (NCEs), is currently one of the most significant difficulties facing modern pharmacy ,2,3. ThiAmorphous materials, due to a lack of long-range molecular order and, associated with that, higher internal energy, are characterized by higher apparent solubilities, faster dissolution rates, and potentially better bioavailability, when compared to their crystalline counterparts ,13,14,15There are quite a large number of polymers commonly used in drug formulation that can reduce the tendency of amorphous drugs toward recrystallization, e.g., poly(vinylpyrrolidone) (PVP), poly(vinylpyrrolidone-vinyl acetate) (PVPVA), poly(styrene sulfonic acid) (PSSA), (hydroxypropyl)methyl cellulose (HPMC), hypromellose acetate succinate (HPMCAS), soluplus (SOP), poly (PVA), and polyethylene glycol (PEG) ,29,30,31On a large scale, ASDs are usually produced using either spray drying (SD) or hot melt extrusion (HME) ,39,40,41The goal of this work was to check the efficiency of four well-known polymers: Kollidon 30 (K30) , Kollidog) of the chosen polymers, its impact will also be discussed. The thermal and viscoelastic properties of POS-based compositions, possessing various polymer content, were investigated to achieve some of the aforementioned goals. Next, the selected concentrations of each drug–polymer composition were extruded. Finally, the thermal properties, long-term physical stabilities, water solubilities, and dissolution rates of the obtained extrudates were investigated. The ultimate aim of this comparative study was to provide general guidance for the screening of polymers suitable for HME.The most important aspects which were checked in this paper were: (i) the amorphous POS stabilization efficiency, (ii) the possibility of manufacturing ASDs by HME, (iii) drug loading, (iv) improvement of the API’s aqueous solubility, and (v) modifications in its dissolution rate. It is worth noting that selecting these polymers allows the assessment of the usefulness of different types of polymers in forming ASDs. As summarized in Posaconazole -5-tetrahydro- 5--3-furanyl]methoxy]phenyl]-1-piperazinyl]phenyl]-2-[-1-ethyl- 2-hydroxypropyl]-2,4-dihydro-3H-1,2,4-triazol-3-one, Wuhan ChemNorm Biotech Co., Ltd., Wuhan, China) was used as a model drug. Two amorphous polymers, i.e., Kollidon K30 , and Kollidon VA64 ), both from BASF, Ludwigshafen am Rhein, Germany, and two semi-crystalline polymers, Kollicoat IR Grafted Copolymer, BASF, Ludwigshafen am Rhein, Germany), and Parteck MXP , Merck KGaA, Darmstadt, Germany) were used as polymeric matrices for ASD. The physical mixtures, containing crystalline POS and one of the listed polymers in concentrations from 10 to 70 wt.% of the polymer, were prepared by co-mixing the appropriate amount of the materials inside an agate mortar. The binary systems, containing an amorphous form of POS, were obtained after the physical mixture’s melt quench. The water used in all tests was produced by an Elix 15UV Essential reversed osmosis system . All other reagents were of the analytical grade.The thermal stabilities of both neat POS and the employed polymers, as well as their physical mixtures, were investigated by a Mettler TG 50 thermogravimetric analyzer  linked to a Mettler MT5 balance . The powder, in open aluminum pans, was placed in a furnace under nitrogen purge (60 mL/min), and heated at 10 K/min. Degradation of the sample was determined in the temperature range from 303 to 723 K, by the weight loss percentage. The data obtained from TGA are attached in the e system , equipped with an HSS8 ceramic sensor, 120 thermocouples, and the liquid nitrogen cooling accessory. The measuring device was calibrated for temperature and enthalpy using zinc and indium standards. The samples were placed in an aluminum crucible (40 μL). Both the initial, i.e., neat crystalline POS, as well the binary drug–polymer systems, containing crystalline API, were heated up from 298 to 453 K (in the case of the systems with K30 or KVA) or to 488 K (in the case of the systems with PXP), or to 503 K (in the case of the systems with KIR), with a rate of 10 K/min. Systems containing amorphous POS were heated up from 298 to 468 K (in the case of systems with K30 and KVA) or to 488 K (in the case of systems with PXP), as well as from 183 to 503 K (in the case of systems with KIR), with the same rate. The thermal properties of the as-obtained, and long-term stored, extrudates were investigated in a few ways. The samples were measured from 278 to 488 K (when containing amorphous polymers), or to 503 K , (i)without an annealing procedure prior to the heating step, with a rate of 10 K/min, through the standard DSC technique, (ii) with a 30 min annealing procedure at T = 373 K prior the heating step, with a rate of 10 K/min, through the standard DSC technique, and (iii) with a 30 min annealing procedure at 353 K (in the case of systems containing KVA), or at 323 K (in the case of systems containing PXP and KIR), prior to the heating step, with a rate of 10 K/min, through the standard DSC technique. It needs to be pointed out that, just before each experiment, the extrudates ) were minced in the agate mortar. The beginnings of the exothermal and endothermal peaks were identified as the crystallization and melting temperatures, respectively. At the same time, the glass transition temperature (Tg) was determined as the midpoint of the heat capacity increment. Each measurement was performed in triplicate (n = 3). Average values and standard deviations (SDs) were calculated.The thermal properties of all investigated POS-based binary systems were measured using the Mettler Tolledo STARThe viscoelastic properties of binary drug–polymer ASDs, containing 50, 60, or 70% KVA polymer, or 40, 50, or 60% K30 or KIR or PXP polymer, were measured utilizing an ARES G2 Rheometer, using an aluminum parallel plate geometry (diameter = 25 mm); the gap was 1 mm. The temperature sweep measurements were conducted at a constant frequency of 1.5915 Hz, and the strain was within the linear viscoelastic region. The samples, in the form of amorphous solid dispersions, were cooled down (i) from 443 to 393 K for POS + KVA systems, (ii) from 453 to 393 K for POS + K30 and POS + KIR systems, and (iii) from 473 to 393 K for POS + PXP systems. A frequency range from 0.016 to 7.958 Hz was utilized.®, Skawina, Poland), equipped with an air-cooled conveying belt . The mixtures of POS and matrix-forming polymers, i.e., K30, KIR, and PXP, in a 50:50 mass ratio, and a mixture with KVA in a 30:70 mass ratio, were extruded through a 1.75 mm die at 50 rpm screw speed. We used standard parallel, corotating, fully intermeshing screws, with three kneading zones, to mix the API and polymer properly. The detailed configuration of the screws was presented in our previous study [HME processes were performed using a 40D, 12 mm corotating twin-screw extruder  ratio, at a flow rate of 1.5 mL per minute. The column temperature was maintained at 313 K. The injection volume was equal to 20 μL. The total run time was 3 min. The retention time of POS was 1.8 min. POS detection was carried out at λ = 262 nm. Linearity was confirmed within the concentration range of 7.8–54.4 μg/mL (R2 = 0.9997). Each measurement was performed in triplicate (n = 3). Average values and standard deviations (SDs) were calculated.An Agilent 1200 Infinity , equipped with a diode array detector and a Kinetex C18 reverse phase LC column Kinetex C18 , were used for analyses. The mobile phase consisted of acetonitrile and a 15 mM potassium dihydrogen phosphate solution (pH = 4.7), ran in an isocratic elution in 55:45 , and filtered through a nylon 0.22 µm KX syringe filter (Kinesis). The samples were analyzed in triplicate using the HPLC method. The reported data represent the averages of three series of measurements with standard deviations (SD).An excess of crystalline POS, amorphous POS (prepared by quench cooling), milled extrudates, and physical mixtures of POS with polymers  were dispersed in 2 mL of distilled water (pH = 5.46). The suspensions were shaken at 400 rpm, at ambient temperature (approx. 295 K), for 48 h, using the Unimax 1010 orbital shaker , until equilibrium was reached. The rotational speed was selected to achieve proper mixing of the suspension and to avoid sedimentation, which can limit the dissolution. The samples were centrifuged at 3600× n = 3). Average values and standard deviations (SDs) were calculated.The dissolution of POS from extruded polymer matrices was performed in the DFZ 60 flow-through apparatus (USP Apparatus 4), with an HKP 60 pump . The extruded material was milled with a Tube Mill 100 . The 100–200 µm particle fraction was used in dissolution studies, to avoid the impact of particle size on dissolution profiles. A single 5 mm glass bead was placed at the bottom of the large flow-through cell for tablets and capsules, and the cell cone was then filled with 2 mm glass beads to achieve constant and laminar dissolution medium flow. The milled extrudates were placed on top of the glass beads and covered with another batch (2 g) of 2 mm glass beads, to prevent the powder from floating and sticking together. A constant 16 mL/min flow of water (pH = 5.46), maintained at 310 K, was used in an open-loop configuration. The samples were collected at predetermined timepoints, and the concentration of POS was assayed by HPLC. All dissolution studies were carried out in triplicate  at 300 rpm. Afterward, the samples were filtered through a nylon 0.22 µm KX syringe filter (Kinesis), and the amount of POS was determined by HPLC.In the first step of our studies, the impact of the chosen polymers  on the melting temperature of POS was investigated. For that purpose, non-isothermal DSC experiments of the physical mixtures were performed. During these experiments, the samples were heated at a rate of 10 K/min. All compositions were investigated at 298 K, while the final temperature depended on the polymer employed. For compositions containing K30 or KVA, the sample was heated up to 453 K. In the case of the systems with PXP or KIR, the final temperature was equal to 488 or 503 K, respectively. Each sample was investigated in triplicate. The representative DSC traces are summarized in As can be seen, the DSC thermogram of neat crystalline POS is characterized by two endothermal processes, which correspond to the melting of the II and I polymorphic forms of this active substance. On the DSC traces of POS + K30 and POS + KVA, one can notice as many as three, while on the DSC traces of POS + PXP and POS + KIR, one can notice four endothermal events. Looking from low to high temperatures, the first endothermic process, in all investigated systems, reflects water evaporation, the second (at ca. 408 K) and the third (at ca. 440 K) events are associated with the melting of the II and I polymorphic forms of POS, respectively. The fourth endothermic process, which is visible only for systems with PXP and KIR, is associated with the melting of the crystalline fraction of the employed semi-crystalline polymers.It is worth noting that, in the case of the POS-based systems containing amorphous polymers , the melting temperature decreases with increasing polymer content. While in compositions containing the semi-crystalline polymers , the lack of a significant depression of the drug melting point is noted. Since depression of the melting point only manifests in miscible drug–polymer combinations, it is reasonable to assume that POS is immiscible with the semi-crystalline polymers used. It is well known that, if the API and polymer are immiscible, melting point depression will not occur, because the presence of the excipient does not alter the chemical potential of the active substance. The possible lack of miscibility is not the only drawback of semi-crystalline polymers employed in POS-based ASDs. Another disadvantage of these polymers is that, due to a higher melting point than that of neat POS, to disperse the API in the polymer matrix utilizing HME molecularly, such systems require heating of the API to above its melting point. Usually, HME polymers must lower the drug’s melting point, to reduce the possibility of thermal decomposition. Thus, when assessing the effectiveness of the employed polymers in lowering the POS melting temperature , they can be ranked as follows: Kollidon VA64 > Kollidon 30 > Parteck MXP > Kollicoat IR.Evidently, the reduction in the API melting point by the polymer is not the only important parameter that should be considered when designing ASDs. Therefore, to (i) estimate the tendency of the employed polymers to form miscible systems with POS, and (ii) assess how the excipients improve the physical stability of the API, the systems were quench-cooled in situ, using DSC, and the glasses obtained in that way were heated up. The quench cooling procedure was performed at a rate of 20 K/min, from a starting temperature of: (i) T = 448 K for systems containing POS and K30 or KVA, (ii) T = 468 K, with 2 min annealing, for POS and PXP systems, and (iii) T = 503 K, as well as T = 448 K with 5 min annealing , for POS and KIR systems. Besides the systems containing KIR, where the cooling procedure was taken down to T = 183 K, the drug–polymer systems were cooled down and reheated from T = 298 K. Similar to the previous studies, all samples were investigated in triplicate. The representative DSC traces of quench-cooled binary compositions are presented in g = 334 K. During further heating, with a rate of 10 K/min, one might note the exothermal process associated with cold crystallization, followed by an endothermal event reflecting melting of the recrystallized part of the API. Based on this information, POS might be classified as a class II material, according to the approach proposed by J. A. Baird [As can be seen, the DSC trace of neat amorphous POS reveals a glass transition event at TA. Baird . These mg = 443, 376, and 341 K, respectively. In the case of KIR, the situation is more complex, since two Tgs were registered—the first at Tg1 = 217 K, and the second at Tg2 = 319 K  can be noted, proving the semi-crystalline nature of these polymers. It is worth noting that all of the above-determined Tg and Tm values agree well with the literature data.The thermograms of the neat, dry polymers K30, KVA, and PXP, shown as gray lines in g2), which, in systems with lower polymer concentrations, might be possibly covered by the crystallization exotherm  can be clearly noted. At the same time, these thermograms reveal the second glass transition event (Therm see a. This rLack of phase separation was noted in the systems containing POS and KVA (amorphous copolymer). As seen in gs of API and polymer. The glass transitions’ heat capacities (ΔCp) vary depending on the drug–polymer concentration. It is also worth noting that, at all investigated concentrations of the system containing PXP, one can notice that POS recrystallizes. This is manifested by the presence of the exothermal process in the vicinity of 400–440 K on the DSC thermograms. This process is followed by two overlapping endothermal events, reflecting the melting of POS and the crystalline fraction of the polymer. Because of the lack of influence of both materials on each other, described above, is clear , one can expect that POS and PXP are not miscible.The last POS-based composition investigated in this work contains KIR—the semi-crystalline copolymer. As seen in g for each system are also presented. The dependencies have been calculated based on the Gordon and Taylor approach [w1 and w2 are the weight fractions of each component, and gT1 and gT2 correspond to the glass transition temperatures of each component. gT is the glass transition temperature of the mixture, while K is a measure of the interaction between the components, and can be defined as follows:To better visualize how the employed polymers modify the glass transition, the onset of crystallization, and the melting temperature of POS, these data are summarized in approach ,61:(1)TgpC1 and ΔpC2 in Equation (2) denote the change in heat capacity at the glass transition temperature of each component .ΔFrom all of the data that has been collected so far, it appears that KVA is the best polymer to create ASD with POS. This amorphous copolymer provides the highest physical stability of the amorphous form, easily forms a homogeneous system with the API, and gives the possibility of employing the lowest production temperature, compared to the other polymers. K30 ranks second, since it can reduce the melting point of POS and stabilize the API when a higher polymer content is employed. Of course, these improvements are not as impressive as in the case of KVA. PXP ranks third, because it does not facilitate amorphous POS’s recrystallization as well as KIR. However, both these semi-crystalline polymers display a lack of (i) homogenization with POS, (ii) significant improvement of the API’s physical stability, and (iii) the depression of POS’s melting point. Based on that, one can conclude that amorphous polymers are undoubtedly better stabilizers of amorphous APIs than semi-crystalline polymers. In the following research stage, the rheological characteristics of the chosen binary systems were examined, to ascertain whether or not KVA would still be the best excipient, offering the appropriate viscosity for producing POS-based ASDs by HME.In this section, the rheological properties of neat POS, and its ASDs containing 50, 60, and 70% of KVA, and 40, 50, and 60% of K30, PXP, or KIR, were investigated, for the selection of one suitable for HME processing temperatures. Temperature sweep measurements were performed at fixed frequencies (0.016–7.958 Hz) for that purpose. These experiments were performed at temperatures (a) from 473 to 393 K (ΔT = 10 K) for systems with neat POS, (b) from 443 to 393 K (ΔT = 10 K) for systems with KVA, (c) from 453 to 393 K (ΔT = 10 K) for systems with K30, (d) from 473 to 373 K (ΔT = 10 K) for systems with PXP, and (e) from 453 to 403 K (ΔT = 10 K) for systems with KIR. The representative data (for neat POS and its systems with KVA) are presented in Due to the significantly lower drug loading in systems with KVA, compared to the other compositions, these results might modify the rankings presented in the previous section, of the polymers’ effectiveness in forming the best POS-based ASD. A high drug loading, with simultaneous high physical stability, makes POS + K30 a potentially better system than POS + KVA. The compositions employing the semi-crystalline polymers—PXP and KIR—also required less polymer  than KVA to ensure an appropriate viscosity of POS. These polymers, however, do not improve the physical stability of amorphous forms of POS. A drawback of the employment of PXP, KIR, and even K30 in formulations with POS worth noting is, the lack of complete homogeneity (described in the last section) or miscibility, as in the case of POS + PXP. However, it is well known that the sample preparation method might impact its homogeneity. Thus, we decided to prepare the extrudates  of the investigated API + polymer systems in the concentrations that had provided the viscosity values most appropriate for HME, i.e., POS + 50% K30, POS + 70% KVA, POS + 50% PXP, and POS + 50% KIR. The main aim of these efforts was to check whether it was possible to obtain better homogeneity of the investigated systems, compared to the method based on the quench-cooling of the physical mixtures.A 40D, 12 mm corotating twin-screw extruder, with an air-cooled conveying belt, was used to produce POS-based extrudates containing the API and different types of polymer. The appropriate amount of polymers for HME was chosen based on the rheological data presented in the previous section, i.e., POS + 50% K30, POS + 70% KVA, POS + 50% PXP, and POS + 50% KIR. HME of systems containing K30, KVA, and KIR went smoothly, confirmed by low torque values and engine loads registered during extrusion, i.e., below 3.5 Nm and lower than 50%, respectively. The process outputs for all systems are given in It has to be noted that the HME process of the POS + PXP system was troublesome. During this process, the melt viscosity was frequently changing, which can be connected to the changes in the ratio between API and PXP. At the end of the process, a melt with fluid-like viscosity leaked from the extruder die. This was probably the melted fraction of API, in which a lack of polymer appears—the phase separation. Our suspicion was confirmed by the POS’s content analysis, indicating that the concentration of API in the extrudates with PXP was significantly lower than the employed POS input.a) was equal to 373 K. This temperature, however, is lower than the system’s Tg only for compositions containing the K30 polymer—in other cases, during the annealing step, samples were stored at the supercooled liquid region. Thus, to dry the extrudate containing KVA, PXP, and KIR, also at T < Tg, additional measurements (the third type of experiment) were performed. These experiments included 30 min sample annealing, at Ta = 353 K for the system containing KVA, or at Ta = 323 K for systems containing PXP or KIR, prior to the main heating step. The obtained DSC thermograms are compared in To investigate the thermal properties of freshly prepared extrudates and those that were stored for up to 120 days, a series of experiments was performed, utilizing standard and temperature-modulated DSC techniques. Extrudates were stored in a glove box at T = 298 K and RH = 25%. Just before all the calorimetric studies, extrudates were minced in the agate mortar. Next, the powdered materials were measured in a few ways. One type of experiment involved heating the tested material, to investigate the sample ‘as obtained’. Such experiments were performed utilizing both the standard DSC technique and TMDSC. The second and third type of experiments included a 30 min annealing step before the standard DSC heating procedure. The annealing aimed to dry the sample. In the second type of experiment, the annealing temperature (TThe POS-based extrudates containing the amorphous polymers (50 % of K30 or 70% of KVA) display similar behavior, thus, will be described simultaneously. Fresh, minced samples are characterized by being slightly covered by the water-evaporating endotherm, a glass transition event. During the storage time, the systems take water in, therefore, DSC thermograms measured after 30 or more days show only one broad endothermal peak, associated with the evaporation of the absorbed water. The DSC curves of the quenched, or dried, extrudates, and their reversible signal obtained from TMDSC, display only one clear glass transition event, proving that by employing the HME technique, both polymers in the chosen concentrations form homogeneous compositions. The lack of exotherm following crystallization, and POS’s melting endotherm on each thermogram, indicates that POS, in composition with either K30 or KVA, is physically stable for a minimum of 120 days of storage at T = 298 K and HR = 25%. As the images presented on the right-hand side of To the unaided eye, a POS-based extrudate that has been melt-quenched, and should contain 50% semi-crystalline PXP polymer, appears more physically stable than its quenched physical mixture. This is because the DSC thermogram of this sample displays a less pronounced exothermal event associated with the recrystallization process, as well as the melting endotherm reflecting the API melting, when comparing it to the thermal events visible on the DSC thermogram of the quenched physical mixture. In the beginning, this might be confusingly associated with better physical stability, connected with better miscibility. However, considering that the phase separation between POS and PXP exists, and the lower-than-expected content of API in the extrudate was proved, the lower enthalpy of fusion of POS should rather be assigned to the lower API content. Due to the lack of miscibility of POS and PXP, we will not discuss the impact of storage time on the extrudate’s physical stability. Nevertheless, it needs to be stressed that, even after 120 days of storage, the vast majority of the API remains still amorphous in this formulation. Such a conclusion is based on the clearly visible glass transition of POS, in both non-dried and dried long-term-stored extrudates.g, located at 238 K, originates from the small fraction of regions in the polymer displaying a low Tg. The second glass transition takes the averaged value of the POS’s Tg and the second glass transition  of KIR polymer. The recrystallization process is associated with the devitrification of the API from the system. Since its enthalpy was significantly different for samples annealed at T = 373 K, such experiments were abandoned after 35 days. The visible reduction in the crystallization enthalpy was associated with the recrystallization of POS, that proceeded during the annealing (T = 373 K) step. First, i.e., located at a lower temperature, the melting endotherm reflects the POS melting, while the second endothermal event, which is noticeable at ca. 470 K, originates from the melting of the crystalline fraction of KIR. As can be seen on the images presented on the right-hand side of Interestingly, besides the highest tendency toward recrystallization noticeable during non-isothermal, calorimetric experiments, the extrudate containing POS + KIR maintained its amorphous form for at least 120 days, when stored at T = 298 K and RH = 25%. The thermograms of extrudates containing POS + semi-crystalline polymer KIR are characterized by the presence of as many as six thermal events. Looking from low to high temperatures, one can distinguish: two glass transition events, a water evaporation endotherm, a crystallization exotherm, and two melting endotherms. The barely seen TThe aqueous solubility and dissolution measurements of the POS-based extrudates show the differences between the solubilizing efficacy of the utilized polymers. The solubility of crystalline POS was below the detection limit for the analytical method (LOD = 0.02 µg/mL). The measured solubility values obtained for the physical mixtures and extrudates are presented in Excluding the POS + PXP system, the best solubility improvement was achieved using K30—an amorphous polymer. It was 7.1 times higher in comparison with the composition containing crystalline POS. Despite the high amount (70%) of the KVA in the POS + KVA system, the solubility, and its improvement, were smaller than those of K30.The dissolution studies were performed in water, which creates unfavorable dissolution conditions for POS, but it was a differentiating dissolution medium. The dissolution profiles are presented in All properties investigated in this paper are summarized in In this paper, the impacts of four different polymers (Kollidon K30 (K30), Kollidon VA64 (KVA), Parteck MXP (PXP), and Kollicoat IR (KIR)) on the physical stability, extrudability, aqueous solubility, and dissolution rate of the amorphous form of posaconazole (POS) were investigated. The aforementioned polymers represent four groups of polymers: (i) K30—amorphous homopolymers, (ii) KVA—amorphous heteropolymers, (iii) PXP—semi-crystalline homopolymers, and (iv) KIR—semi-crystalline heteropolymers. The performed studies prove that amorphous polymers (KVA and K30) are undeniably better excipients than semi-crystalline polymers (KIR and PXP), for formulations containing amorphous POS. These types of polymers guarantee homogeneity and high physical stability when mixed with the POS. The POS-based extrudates obtained with K30 and KVA are transparent, and show between ca. 4 to 7 times better POS aqueous solubility than their physical mixtures . The main differences in the efficiencies of the tested amorphous polymers in creating POS-based ASDs suitable for HME, result from the ease of creating a homogeneous system and drug loading. Employing K30, a higher amount of drug can be used to obtain a melt viscosity suitable for HME, compared to utilizing KVA, while the KVA guarantees better homogeneity. Moreover, POS K30 extrudate is characterized by uniform shape and good mechanical strength, which leads to the conclusion that it may be utilized as a filament in fused deposition modeling 3D printing. The main disadvantages of the utilized semi-crystalline polymers are, the lack of POS melting point depression in their systems, and the lack of complete homogeneity or even miscibility, as in the case of the POS + PXP system. Despite the worst improvement in solubility, the KIR—the representative heteropolymer—was ranked as the third excipient for the formulation of POS-based ASDs. This is because POS extruded with KIR is characterized by an appropriate content, and reveals completely amorphous form, for at least 120 days of storage at standard storage conditions. PXP cannot fully protect POS from recrystallization and form a miscible system with it. Nevertheless, in the future, it would be worth exploring the mechanism responsible for this semi-crystalline homopolymer improving POS’s aqueous solubility.


In this study, we examined the sub-acute toxicity of quercetin and ferulic acid and evaluated their effects on protein, cholesterol, and estrogen levels in vivo. Six groups of female Wistar rats were fed by gavage. The first and second groups represent the positive (Clomiphene citrate 10 mg/kg) and negative (NaCl 0.9%) control groups, while the other groups received quercetin and ferulic acid at doses of 5 and 10 mg/kg/day for 28 days. The sub-acute toxicity was monitored by examining the weights, biochemical parameters , and histological changes in the kidneys and liver of the treated animals. Furthermore, the in vivo estrogenic effects were studied in terms of the serum and ovarian cholesterol levels, serum estradiol, and uterine proteins. Finally, Docking studies were conducted to evaluate the binding affinity of quercetin and ferulic acid for alpha and beta estrogen receptors. Results showed that both compounds were devoid of any signs of nephrotoxicity or hepatotoxicity. Additionally, quercetin and ferulic acid caused significant estrogenic effects evidenced by an increase of 8.7 to 22.48% in serum estradiol, though to a lesser amount than in the reference drug-treated group (64.21%). Moreover, the two compounds decreased the serum cholesterol levels (12.26–32.75%) as well as the ovarian cholesterol level (11.9% to 41.50%) compared to the negative control. The molecular docking in estrogen alpha and estrogen beta active sites showed high affinity of quercetin  and ferulic acid  to these receptors. This study provides promising insights into the potential use of quercetin as a therapeutic agent for the management of female fertility issues. Infertility is diagnosed when a couple is unable to conceive within six months for women over 35 and one year for women under 35. The inability to carry a pregnancy to term and deliver a living child is another definition of infertility . In the A variety of medical treatments are available for infertility, encompassing the utilization of fertility medications to induce “superovulation,” which involves the development and release of multiple eggs during each ovulatory cycle, along with intrauterine insemination and advanced assisted reproductive technologies . NeverthSenecio biafrae, Petroselinum sativum, and Lavandula officinalis [While there are many pharmaceuticals available to treat female infertility, there is increasing interest in alternative and complementary therapies, such as plant-based remedies. Herbal medicines have been used for centuries to treat reproductive problems, and there is evidence suggesting that certain plants can effectively treat infertility in women ,9. This icinalis ,11,12. Iicinalis . Phytoesicinalis  and beneicinalis . These cicinalis ,17.Petroselinum sativum [Lavandula officinalis [In our previous studies, we investigated the estrogenic effect of  sativum  and Lavaicinalis  extractsNumerous studies have demonstrated the implications of phenolic compounds from medicinal plants on the regulation of reproductive functions ,18. The In this study, we explored the estrogenic potential of quercetin and ferulic acid in female Wistar rats by assessing their effects on serum and ovarian cholesterol levels, serum estradiol, and uterine proteins. In addition, an in silico study was performed to assess the affinity of both quercetin and ferulic acid for estrogen receptors.Throughout the 28-day treatment period, all animals exhibited significant weight gain . HoweverTo assess the effect of the different treatments on the renal and hepatic functions, we analyzed the relevant serum parameters, mainly ALAT, ASAT, urea, and creatinine. Our findings demonstrated that the administration of quercetin and ferulic acid at doses of 5 and 10 mg/kg did not result in any adverse effects on hepatic parameters, as evidenced by ALAT A and ASAThe examination of tissue samples treated with quercetin and ferulic acid under a microscope revealed no histological abnormalities in the liver and kidneys. Specifically, there were no observable changes or histopathological abnormalities in the structures of the central vein, hepatocytes, and hepatic sinusoids, indicating the absence of liver toxicity .Likewise, the examination of histological sections of the kidneys revealed that none of the administered doses had an impact on renal structures, including the glomerulus, proximal convoluted tubules, and distal convoluted tubules .p < 0.01), with comparable performance to the standard drug, clomiphene citrate  (p < 0.001), compared to the negative control group (< 0.001) A. Similaol group B. In conAfter 28 days of treatment, no significant difference was noticed in the uterine protein levels A. MoreovThe results of the histological sections of the ovaries  treated The glide gscore, glide emodel, and glide energy for quercetin in estrogen alpha’s active site were −10.444, −82.345, and −54.171 kcal/mol, respectively. Moreover, quercetin showed in the estrogen beta receptor active site a glide gscore, glide model, and glide energy of −10.662, −74.309, and −51.529 kcal/mol .In the active site of estrogen alpha, the ferulic acid showed a glide gscore, glide emodel, and glide energy of −6.377, −39.698, and −27.718 kcal/mol. Ferulic acid showed in the active site of estrogen beta a glide gscore, glide model, and glide energy of −6.3, −38.943, and −27.308 kcal/mol .When quercetin was docked into the active site of estrogen alpha, it was observed that it formed four hydrogen bonds with GLU 305, ARG 346, LEU 339, and HIE 475 residues, along with a Pi–Pi stacking interaction with residue PHE 356. Furthermore, the docking of quercetin in the active site of Beta estrogen showed the formation of the same bonds with the same residues .In the active site of estrogen alpha, the docking of ferulic acid presented the formation of a single hydrogen bond with the residue HIE 475, whereas the docking of this molecule in the active site of estrogen beta showed the formation of one hydrogen bond with residue HIE 475 and one salt bridge with residue AGR 346 .In this study, we examined the sub-acute toxicity of quercetin and ferulic acid and evaluated their effects on protein, cholesterol, and estrogen levels in vivo. We provided evidence that both quercetin and ferulic acid are devoid of any toxicity towards the two key organs of the metabolic processes and detoxification, namely, the liver and kidneys . In addiThe proliferation of human breast cancer MCF7 cells is stimulated by ferulic acid in a concentration- and estrogen-receptor-dependent manner. This effect is associated with the upregulation of HER2 and estrogen receptor α expression .The fact that cholesterol acts as the main precursor for hormones and steroids is widely recognized ,29. HereEstradiol is a key hormone in female reproductive function. In our study, we observed a significant rise in estradiol in the quercetin group at the dose 10 mg/kg compared to the negative control group. The presence of estradiol-inducing secondary metabolites in plants, such as lignans, daidzein, and genistein, was proposed as a potential explanation for the observed similar induction ,12. The Phytoestrogens are a group of naturally occurring compounds found in plants that have a similar structure to the estrogen hormone . The mecQuercetin and ferulic acid showed a vasodilating effect of estrogen, which is partly linked to its ability to improve the bioavailability of nitric oxide (NO) ,43, whicIn addition to their capability to bind to estrogen receptors, phytoestrogens exert other biological effects that are independent of these receptors. These effects include the activation of serotoninergic receptors, IGF-1 receptors, and the ability to bind free radicals. They can also induce DNA methylation and affect various intracellular regulators such as tyrosine kinase, cAMP/protein kinase A, cGMP/NO, phosphatidylinositol-3 kinase/Akt, and MAP kinases . Moreover, they have the capacity to impact transcription factors like NF-kappaB and DNA topoisomerase activities, histone modification, RNA expression, and various intracellular regulators associated with cell cycle and apoptosis .Estrogen receptors play a crucial role in the regulation of female infertility. Estrogen is a hormone that is essential for the development and function of the female reproductive system. Estrogen receptors are proteins that are located in various tissues of the body, including the uterus, ovaries, and hypothalamus . In the In this work, an in silico investigation was conducted to examine the affinity of quercetin towards estrogen alpha and beta receptors. Interestingly, we found that quercetin exhibited remarkable binding activity of −10.444 and −10.662 kcal/mol, respectively. Likewise, ferulic acid, a compound that has been reported for its role in female infertility ,51, alsoThe ability of quercetin and ferulic acid to induce apoptosis in different cancer cell lines has been extensively studied ,53,54. QThis study shed light on the promising insights regarding the potential use of quercetin as a therapeutic agent for the management of female fertility issues through its interaction with estrogen receptors. Recent studies have unveiled its ability to modulate estrogen receptor signaling pathways, suggesting its potential to regulate estrogen levels and improve reproductive outcomes. By targeting estrogen receptors, quercetin may play a crucial role in maintaining hormonal balance, promoting follicular development, and enhancing ovulation. Furthermore, its anti-inflammatory effects could alleviate reproductive disorders associated with inflammation. While further research is necessary to fully elucidate the mechanisms involved, these promising findings pave the way for potential therapeutic applications of quercetin in the realm of female fertility, offering hope for improved management and treatment of reproductive challenges.Ferulic acid and quercetin were obtained from Sigma Aldrich .Wistar rats with an average weight of 192 ± 18 g were used for the in vivo experiments. Animals were bred in the animal house of faculty of science in Fez, Morocco. The animals were raised in the animal facility at the Faculty of Science situated in Fez, Morocco, where the temperature was maintained at 22 ± 2 °C and a D/N cycle of 12 h/12 h was used. Animals were provided with unrestricted access to both food and water. The protocol for animal care and use has been submitted to the institutional ethics committee in accordance with the European Community Directive EEC/86/EEC .Quercetin and ferulic acid were dissolved in distilled water and orally administered to rats . TreatmeTo assess the sub-acute toxicity of quercetin and ferulic acid, the guideline no. 407 was followed . The weiTo evaluate the ovarian stimulation effect of quercetin and ferulic acid, the animals were dissected after 28 days of treatment. Initially, the uterus and ovaries were weighed and pulverized in a NaCl (0.9%) solution. After centrifugation, the resulting supernatants were collected to evaluate the levels of ovarian cholesterol and uterine protein. This assessment was conducted using the enzymatic colorimetric technique with peroxidase (POD) for cholesterol and the biuret method for protein. . The bloThe ovaries were preserved in a 10% formalin solution. Then, histological examination was performed using standard techniques of microscopic observation via staining with hematoxylin and eosin .Estrogens are the key hormones involved in the regulation of fertility. These two hormones are necessary for the cellular responses mediated by estrogen receptor α and β [ α and β . TherefoThe compounds quercetin and ferulic acid were obtained from PUBCHEM in SDF format. The LigPrep tool in the Schrödinger Software program (version 11.5) with the OPLS3 force field was used to prepare the ligands for docking calculations. A total of 32 stereoisomers were generated for each ligand after selecting the ionization states at pH 7.0 ± 2.0 .The crystal structures of estrogen alpha and estrogen beta were obtained from the protein data bank with PDB IDs 1X7B and 1U3Q, respectively. The Protein Preparation Wizard in Schrödinger-Maestro (v11.5) program was used to further refine and prepare the structures. All water molecules were removed, hydrogens were bonded to heavy atoms, methionines replaced selenomethionines, and charges and bond orders were subsequently assigned. The OPLS3 force field was utilized to carry out minimization, resulting in a maximum heavy atom RMSD of 0.30 Å. .An atom from the ligand was chosen, and a default grid box was made. The grid box had volumetric spacing of 20 × 20 × 20. Using the ‘Extra Precision’ (XP) method, the ligand was linked to the grid box produced by the protein. The XP GScore was used for assessing the results .Flexible ligand docking using the Glide module in Schrödinger-Maestro (v11.5) was performed with standard precision (SP). Penalties were given for non-cis/trans amide bonds and the parameters for van der Waals scaling factor and partial charge cutoff of ligand atoms were both established as 0.80 and 0.15, respectively. The assessment of the final scoring was performed on energy-minimized poses, with the utilization of the Glide score to evaluate the outcomes. The top-docked pose with the lowest Glide score value was identified and recorded for each ligand .p < 0.05 was established, and the results were presented as the mean ± standard error of the mean (s.e.m.).The data were analyzed using Graph-Pad Prism 5 software, and the Tukey’s test was employed to compare the different treatments. A significance level of This study offers scientific evidence supporting the potential benefits of quercetin and ferulic acid in addressing female infertility. It highlights their role as potentially safe anti-infertility agents when administered orally to female rats. Remarkably, our findings provide novel insights into the estrogenic properties of quercetin. Hence, conducting comprehensive investigations using cellular and animal models, along with clinical studies, would be crucial in order to elucidate the underlying mechanisms by which quercetin affects biomarkers related to fertility. Moreover, administering higher concentrations would reveal more information about the extent of the compound’s efficiency and safety. Although phytocompounds are credited with numerous health advantages, such as decreasing the chances of developing osteoporosis, heart disease, breast cancer, and menopausal symptoms, phytoestrogens could also induce endocrine disruptions, which implies they may have negative consequences on health. Therefore, special focus should be given to evidence supporting and contradicting the alleged advantages and drawbacks of phytoestrogens. Although this in silico study showed the promising therapeutic potential of quercetin against estrogen receptors, it is suitable to consider other ligands and receptor targets  and follicle-stimulating hormone (FSH)). It is also worth studying the effects of quercetin on the secretion of FSH, LH, and progesterone and thus modulating hormone imbalances and supporting reproductive health. Ultimately, this study highlights the potential therapeutic value of quercetin in addressing female infertility conditions.


BRCA1/2 mutation or deficient in the homologous recombination repair system (HRD) generally benefit from treatment with PARP inhibitors. Some international recommendations suggest that BRCA1/2 genetic testing should be offered for all newly diagnosed epithelial ovarian cancer, along with HRD assessment. Academic tests (ATs) are continuously under development, in order to break down the barriers patients encounter in accessing HRD testing. Two different methods for shallow whole-genome sequencing (sWGS) were compared to the reference assay, Myriad. All these three assays were performed on 20 retrospective HGSOC samples. Moreover, HRD results were correlated with the progression-free survival rate (PFS). Both sWGS chemistries showed good correlation with each other and a complete agreement, even when compared to the Myriad score. Our academic HRD assay categorized patients as HRD-Deficient, HRM-Mild and HRN-Negative. These three groups were matched with PFS, providing interesting findings in terms of HRD scoring and months of survival. Both our sWGS assays and the Myriad test correlated with the patient’s response to treatments. Finally, our AT confirms its capability of determining HRD status, with the advantage of being faster, cheaper, and easier to carry out. Our results showed a prognostic value for the HRD score.High-grade serous ovarian cancer (HGSOC) patients carrying the  Ovarian cancer (OC) is the most lethal gynecological cancer, and the eighth leading cause of cancer-related death among women worldwide ,2; the WThe standard treatment for EOC includes surgery and platinum-based chemotherapy, but BRCA1/2 mutation carriers respond well to platinum-based chemotherapy and (PARP) inhibitor treatment . DiffereThe BRCA1/2 proteins play a crucial role in the repair of double-strand breaks of DNA, by a homologous recombination system. In the HGSOC showing a deficiency in this system, drugs targeting PARP1/2 enzymes have a strong anti-tumor activity, due to a mechanism known as synthetic lethality .It is known that treatment with PARP inhibitors of HGSOC patients carrying a BRCA1/2 mutation led to a prolonged progression-free survival (PFS): nevertheless, some patients without these alterations showed similar benefits from the same treatment .Consequently, the identification of patients deficient in the homologous recombination repair (HRR) system , who could benefit from this treatment, still represents a clinical unmet need . The altThe definition of the overall molecular signatures surrounding the HRD status , could lead to the better selection of patients who benefit from PARP inhibition .Due to the evidence that, in OC and breast cancers, the HRD score defined by telomere allelic imbalances, large-scale transitions, loss of heterozygosity (LOH), and mutational signature 3, showed higher sensitivity to platinum and PARP inhibitors, even in the absence of BRCA1/2 mutations, the implementation of HRD assessment is becoming mandatory for the clinical management of OC patients .Up until today, several works evaluating the different methodologies available for defining HRD status have been published. Nonetheless, there is not yet a unique assay able to determine HRD status of all cancers potentially eligible for treatment with PARP inhibitors.After the results of the PRIMA and PAOLA1 trials, the gold standard for the identification of the HRD status is the commercial assay myChoice CDx  .This test detects the presence of BRCA1 and BRCA2 alterations, along with other parameters  ,11. HoweBRCA1/2 mutations, but the mutational status of these genes is not always determined correctly by commercial tests, with evident discrepancies in methodological comparison works [The major issue is the definition of HRD status, which is related to the type of assay performed. For example, the myChoice CDx assay relies on the presence of the on works .Lately, the MITO  group has published a work on the development of an academic test able to overcome the difficulties due to the logistics- and access-related issues, which are mainly due to the following: (a) the lack of a harmonized NGS infrastructure in Italy, as in other European countries; (b) the high costs of the outsourced assays; and (c) the differences in the score calculated by the available commercial and outsourced assays, with the need for the coming IVD rules to comply within European countries ,13. The The main aim of this work was to compare the academic HRD-MITO assay (aHRD-MITO) as performed using the shallow NGS Roche method , coupled with the bioinformatics algorithm for HRD deciphering , with a The secondary endpoints were the following: (a) the comparison of the analytical performances obtained by R and W sWGS pipelines with the reference assay provided by Myriad ; (b) thehttps://franklin.genoox.com/clinical-db/ accessed on 10 September 2023) as Class 4 (likely pathogenic), was indeed annotated by Myriad as a non-clinically significant finding. We underline the fact that the c.649del variant resulted to be exclusively somatic, with a coverage of 40% in our patient. The definitive classification of this variant is still pending.In patient n.1, Myriad did not report the c.4284dupT BRCA2 alteration that was arbitrarily listed within the “non-clinically significant findings” as pathogenic. This variant, which was also confirmed at germline level, not only in the proband but also in family members suffering hereditary cancers, is indeed reported as Class 5 (pathogenic) in all genomic databases (ClinVar and Varsome). In patient n.4, the BRCA1 c.649del, which is reported by the Franklin database .In patient n.19, Myriad did not identify the BRCA1 c.65T>C mutation that we confirmed also at germline level in this patient. Therefore, the borderline HRD scoring of 41 given by Myriad can be not accurate, due to the missing information regarding BRCA status, while our HRD assessment resulted as to be highly positive.HRD-Deficient (LGA > 20), HRM-Mild (15 < LGA < 19) and HRN-Negative (LGA < 14) [R” and “W” methods are highly correlated to each other, with a Spearman’s rho coefficient of 0.914 (p = 0.000). After patient stratification [p = 0.000).Sequencing data obtained from both sWGS kits were used to classify the samples with the aHRD-MITO scores, based on our in-house bioinformatic pipeline. The scores obtained were used to estimate the level of correlation between the two technologies, as well as to stratify the patients in three groups. Based on the large-scale genomic alteration (LGAs), we identified the following: GA < 14) . We usedGA < 14) . In Tablfication , Cohen’s®. We obtained a Spearman’s rho coefficient of 0.734 (p = 0.000) and 0.699 (p = 0.001) for W and R, respectively, when compared to the Myriad score, corresponding to a substantial agreement. In order to compare the results with the Myriad classification, we used the GIA obtained using the two in-house methods, to stratify the patients into two groups: HRD-Deficient (LGA > 15), and HRN-Negative (LGA < 14)  and 0.483  for R and W, respectively. As expected, the Fisher’s exact test also resulted to be perfectly in agreement with the other statistical tests. The method R showed an overall better level of agreement with the Myriad patients’ classifications. Therefore, method R was used for the further comparisons and data analyses. Finally, we tried to associate our three different scores  with the scores given by Myriad, as shown in Seventeen samples had positive HRD results for cutoff ≥42. In contrast, R and W scores were positive in 16 and 17 samples, respectively. The PPA was 94% for R and 88% for W, compared to myChoice. Cohen’s kappa was 0.608  univariate analysis utilizing three different LGA thresholds  are shown in It is noteworthy that our scores significantly correlated with PFS: in fact, patients with the highest HRD scoring showed the longest mean PFS (95.04 months), as compared to the mild (39.97 months) and negative (20.73 months) ones.In this study, we carried out two methods of shallow whole-genome sequencing (R and W) to identify genomic instability and assess the HRD status in 20 ovarian cancer samples, nineteen classified as HGSOC and one as endometrioid, all collected before administering drug treatment.When evaluating the R and W aHRD-MITO assays, both shallow NGS methods showed a high correlation with each other, and a complete agreement. Even when compared with the score provided by Myriad, a substantial agreement was found.Nevertheless, when we stratified the patients into HRD-Deficient and HRN-Negative groups for the comparison with the Myriad classification, the R methods showed an overall better level of agreement, and were therefore used to set up the subsequent analysis.BRCA1/2 status or the molecular signatures (SBS3 and SBS8) [Based on our results, we were able to assign as negative (HRD-N) those samples below the Myriad score < 42, as HRD-mild those corresponding to Myriad score values between 43 and 55, and as HRD-Deficient those with a Myriad score above fifty-six. These results are representative of the significant level of agreement found in our samples obtained using the same bioinformatics pipeline in the two different sWGS assays. It is noteworthy that, although the aHRD-MITO scoring was assessed without evaluating the nd SBS8) , we wereFurthermore, when the correlation of the HRD classification of the “R” method with progression-free survival (PFS) was performed, patients classified as HRD showed a significantly higher probability of survival, with approximately 95 months of mean PFS, as compared to those classified as HRM (PFS = 39.97) or HRN (20.73). These findings are in agreement with results obtained by other authors in similar contexts .BRCA1/2 mutations can contribute equally to the HRD feature [BRCA1/2 somatic testing can also fail to detect pathogenic variants with highly consolidated NGS pipelines [BRCA1/2 mutation could show an HRD status which is dependent on other mechanisms of HRR impairment (RAD51 loss or methylation of BRCA1 promoter).It is important to underline the fact that, although the BRCA1/2 mutational status can contribute to an improvement in different bioinformatics pipelines, it has been recently established, as not all  feature , and BRCipelines . TherefoBRCA1/2 germline/somatic testing should be implemented, to enable optimal and timely treatment decisions on maintenance therapy, as well as for testing patients on whom the HRD test will not be evaluable [Nevertheless, as recently reported, the co-testing of HRD and valuable .Furthermore, it has recently been shown that the benefit of combo therapy (olaparib and bevacizumab) is particularly high for patients with mutations located in the BRCA1/2 DNA-binding domain, and is also associated with a good or excellent outcome . TherefoMoreover, our data show that HRD testing can assume both a prognostic and a predictive value, since PFS is strictly correlated with the value of the score.Finally, we would underline many important issues coming from our study, which could facilitate, in the near future, the access to this type of test, which is still available only from outsourcing or to different alternative methods, whose reimbursement is still challenging for most of the national and regional health systems ,22.(a)Our assessment is completely independent of the BRCA1/2 results. In this regard, although the guidelines for tumor BRCA testing  have bee(b)Our bioinformatics analysis for GIA scoring was independent of the type of sWGS employed, since both “R” and “W” chemistries resulted to be qualified for the large-scale genomic alteration assessment, providing superimposable results, even when compared to the Myriad assay. Thus, our data confirm the utility of using the sWGS pipeline in assessing HRD status.(c)Costs for genomic testing are becoming an important barrier for patients who have some difficulties in covering the costs of this test on their own. Moreover, health systems do not have the capability to sustain financial coverage of overall genomic assays. In this regard, the availability of HRD assays that are cheaper and more accessible to patients is an important goal to achieve, in order to facilitate patient pathway and management. In this regard, our HRD assay is (1) easy to carry out; (2) fast, since it takes only four full days to prepare and run forty-eight samples; (3) cheap, since overall costs for our test are close to EUR 500; and (4) implementable by different hospital laboratories participating in the HRD Italian network within the MITO-group.We consider our results to be really exiting, for the following reasons:Our results are in favor of rapid implementation of the HRD test in the clinical setting, particularly when performed under ISO1589 rules, to respond to the quality assurance and IVDR requirements, particularly for laboratory-developed tests, in the limbo of this transition period of IVDR implementation .To achieve this goal, we should also make some efforts to reduce biases related to the pre-analytical level, where tumor purity has been recently shown to be the most critical factor for the determination of the HRD score .As a limit of our study, we can underline the small cohort of patients analyzed: although we performed the test on only twenty samples in three different modalities, our patients were all fully clinically characterized, also for the follow-up. We want to point out that the HRD MyChoce is very expensive, and costs for the NGS reagents (particularly the high-throughput flow cells) are high; not all research groups can cover costs for these analytical comparisons, outside of clinical trials supported by big pharma, as is the case in the present study.Finally, the standardization and harmonization of the entire HRD pipeline is becoming mandatory, in order to guarantee the best testing and patient management and, in particular, to match the ISO15189 and IVDR requirements .® CDx, in comparison with our previously published in-house academic genomic tests of genetic instability (namely aHRD-MITO) and the “W” pipeline [Under a protocol approved on the 29 of June 2023 by the Ethical Committee of Enna “Kore” University (prot. N 12147/2023) 20 high-grade serous and endometrioid ovarian-cancer patients who received surgery at the Department of Obstetrics and Gynecology of Cannizzaro Hospital were retrospectively enrolled, in a consecutive series. Cancer tissue specimens were used to assess the HRD genomic instability score (GIS) evaluated with regulatory-approved Myriad myChoicepipeline . Data coThe mutational status of the 20 enrolled patients was as follows: N = 18 BRCA-mutated ; 1 = carrier of BRCA2 VUS; and 1 was negative for BRCA alterations. The types of alterations are listed in ® Fluorometer 4.0 , and the value was used to convert ng/ul to nM. Two nanomoles of each “W” library was prepared, before pooling all the samples together. The paired-end sequencing reaction was performed on the Illumina NextSeq550 Dx System , loading the pool with a concentration of 1.5 pM and 2% Phix 1.5 pM.Total genomic DNA from formalin-fixed paraffin-embedded (FFPE) tissues was extracted with the QIAamp DNA FFPE Tissue Kit , according to manufacturer’s instructions. Shallow WGS DNA libraries for Illumina sequencing were prepared using two different methods: (1) “R”—the Roche shallow WGS KAPA HyperPlus kit, according to the manufacturer’s instructions , following the same protocol already published ; and (2)Clinical data were extracted from electronic medical records, including age, FIGO stage, tumor grade, histology, chemotherapy, Overall Survival (OS), and Progression-Free Survival (PFS) .As indicated by our AIOM-Guidelines , all thein-house pipeline. At this stage, six independent models spanning the 5-to-1000 kilobase genome window, implemented using the LGA method [GIA was calculated using whole-genome sequencing (WGS) data at low coverage (0.4–0.8×) for each sample, using six different integrated models, encompassing variable sliding windows spanning 5–1000 Kb. Briefly, all fastq files passing quality control were processed and aligned to the hg19 reference genome, using a customized Sarek pipeline ,30, builA method  and a neA method , provideA method .Descriptive statistics were used to summarize the demographic and clinical characteristics of the study population. Baseline characteristics of patients were described using the median and interquartile range for continuous variables and frequencies and percentages for the qualitative variables. The level of correlation between the 2 WGS technologies (R and W) used to calculate the aHRD-MITO scores was established using Spearman’s coefficient. Also, the test of association, Fisher’s exact test, was used. The concordance index was measured using Cohen’s k statistic, with 95% CI. The k statistic was interpreted as <0, indicating no agreement, 0.00–0.20 as slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as an almost perfect agreement. The percentage positive agreement (PPA) among the scores in the entire cohort was also assessed. Overall survival (OS) was measured from date of diagnosis to date of death or last follow-up. The patients who were alive were censored at the date of the last follow-up. PFS was measured from the date of diagnosis to the date of earliest recurrence, progression, or death. Patients were censored on the date of the last evaluation for recurrence or progressive disease. Kaplan–Meier was used to estimate OS and PFS, and results were modeled using the Cox proportional hazards regression as a function of prognostic factors for estimating the hazard ratio, with 95% confidence intervals. All statistical analyses were performed using SPSS software ver. 26.0 .BRCA1/2 testing. Our data can be helpful in the implementation of the HRD assay in routine clinical settings.The present paper shows that, when sWGS is coupled with a powerful and a clinically validated bioinformatics pipeline, HRD can be easily assessed, regardless of


Further investigations of retinal VTI in patients with FD could be helpful to use OCTA as a noninvasive screening and follow-up modality to assess disease progression in affected patients. Vessel tortuosity (VT) is a parameter used to assess retinal involvement in patients affected by systemic diseases such as Fabry disease (FD). In this study, we assessed a retinal VT index (VTI) using optical coherence tomography angiography (OCTA) in a group of patients with FD (FD cohort) compared to a healthy control group (HC cohort). This is a single-center, retrospective study analysis of all consecutive patients with genetically tested and confirmed FD who underwent regular ophthalmological visits from December 2017 to January 2020 at the Department of Ophthalmology at the University Hospital of Zurich, Switzerland. VTI was calculated for each OCTA image and the results were compared between FD and HC cohort. A total of 56 participants, 32  in the FD cohort and 24  in the HC cohort. Classic onset was determined in 18 patients. Overall, mean VTI (±SD) was 0.21 (±0.07). Male patients with classic-onset FD had a significantly higher mean VTI  compared to all other subgroups ( Systemic diseases, including rare genetic diseases, can lead to pathological changes in the retinal vasculature, resulting, for example, in dilated retinal vessels, microaneurysms, neovascularizations, and an increase in vessel tortuosity (VT) ,2,3. Fabry disease (FD) is a rare inherited, X-linked lysosomal storage disorder with an estimated prevalence of between 1:17,000 and 1:117,000 . In patiIn general, the disease is divided into a classic and late-onset phenotype ,16. PatiSo far, numerous direct measurements of systemic vascular function have been established ,22,23. HEpidemiological studies have shown that using the comparison of arterial and venous perfusion, retinal vessel calibers, and retinal arteriolar narrowing, retinal vasculature showed alterations dependent on gender, ethnicity, and age ,25,26,27Retinal imaging allows an accessible way to analyze the vascular network, allowing it to be a time and cost-efficient examination modality ,35. The θ·(LA/LC)). M stands for the average ratio of centerline length to chord length between two inflection points. LA and LC are the length of the centerline (A) and chord (C). N is the number of critical points where the first derivative of the centerline vanishes. SDθ equates the standard deviation of angle differences between lines tangent to each centerline pixel [Over the past decades, several approaches have been assessed to quantify retinal VT using optical coherence tomography angiography (OCTA) with different mathematical models ,40. To one pixel . In this study, we aimed to assess retinal VT in patients with FD (FD cohort) and to compare the findings with a healthy control group (HC cohort), by analyzing a novel VTI using binarized OCTA images of a swept-source device using en face images of the superficial capillary plexus (SCP).This is a single-center, retrospective analysis of all consecutive patients with genetically tested and confirmed FD who underwent regular ophthalmological visits from December 2017 to January 2020 at the Department of Ophthalmology at the University Hospital of Zurich, Switzerland. Written informed consent was obtained before patients’ and probands’ participation in this study. The institutional review board approved the study  and adhered to the tenets of the Declaration of Helsinki [Patients with genetically confirmed FD, which were 16 years old or older, were included in our FD cohort. FD patients with ocular diseases unrelated to FD, including significant lens opacification, current or previous macular and retinal vascular diseases, diagnosis of glaucoma, congenital eye diseases, and high myopia (>4 diopters) were excluded. Non-age-matched healthy subjects, without systemic vascular or ocular diseases, having a best-corrected visual acuity (BCVA) of logMAR 0.1 or better, were included in the HC cohort.®, Nidek Inc., San Jose, CA, USA). IOP measurements were acquired using the same device by Nidek.All study subjects underwent a complete ophthalmological examination, including BCVA, IOP measurement, biomicroscopy of the anterior segment, and funduscopy. BCVA was assessed using glasses with refractory values obtained using an autorefractor  and swept-source OCTA .OCTA volumes of 6 mm × 6 mm × 2 mm centered on the fovea were acquired by a well-trained ophthalmologist (M.W.). Scans with a signal strength of 8 out of 10 or higher were included. Additionally, images with incorrect centration and motion or projection artifacts were excluded. Acquired OCTA scans of the SCP were exported from the PlexElite Device in png format. Based on the detection method of Steger et al., ridge detection was used in Image J  to convert the images into a binary, skeletonized file . The skeletonized image was then imported to MatLab  to perform VTI calculation .Therefore, VT was assessed with a mixed approach to measure vessel length and curvature. For each 6 × 6mm OCTA scan of the SCP, we selected all vessels for analysis. Therefore, all bifurcation and endpoints of the vessels were taken into processing. Vessel centerlines between each pair of bifurcation points were extracted through manual endpoint selection. The vessels’ endpoints were selected by simultaneous visualization of the grayscale and binary images. The grayscale image was used to identify bifurcations, and the binary image was used to locate the bifurcations more accurately with respect to the vessel boundaries and centerline. Afterward, the vessels could be segmented. Each vessel segmentation was defined manually, resulting in an individual VTI for each vessel segmentation in one OCTA image. After calculating the VTI of all vessel segments individually, the mean VTI was calculated per image using the VTI Calculation program created by Khansari et al. ,45.Descriptive statistics are presented as counts and frequencies for categorical data and mean, including standard deviation, for metric variables. p-values. p-values and confidence intervals are adjusted using Tukey’s method.Results are presented as differences in mean VTI with corresponding 95% confidence intervals and p-value < 0.05 is considered significant. Overall p-values correspond to the T-test for means and Chi-squared or exact Fisher’s test. To compare mean VTI between study groups, an ordinary linear regression model was used. Additionally, age was included as a covariate, hence results are adjusted for these variables. All evaluations were carried out using the statistical software R .A In total, we imaged 62 eyes of 62 participants (34 in the FD cohort and 28 in the HC cohort). Two eyes of the FD cohort and four eyes of the HC cohort had to be excluded due to the low quality of the images. Hence, 56 eyes of 56 patients, 32 in the FD cohort and 24 in the HC cohort, could be included for final analysis. The male:female ratio was overall 25:31 (12:20 in the FD cohort and 13:11 in the HC cohort). Overall, the mean age (±SD) was 37.7 (±13.56), 42.3 (±14.4) in the FD cohort, and 31.58 (±9.56) in the HC cohort. Overall, the mean VTI (±SD) was 0.21 (±0.07) in all assessed OCTA images, 0.23 (±0.09) in the FD cohort and 0.22 (±0.09) in the HC cohort, respectively. In the FD cohort, 18 patients had a classic type , and 14 patients had a late onset of the disease . An overview of the demographic data and the mean VTI (±SD) is provided in Comparison of mean and median VTI between FD cohort and HC cohort did not show significant differences.p-value < 0.05, Subgroup analysis showed that male patients with a classic type of FD had the highest mean, median, and individual data points VTI , which was significantly higher compared to every other subgroup  compared to a healthy control group (HC cohort).Regarding vessel selection, we selected all the vessels of the SCP per OCTA image. Vessel centerlines between each pair of bifurcation points were extracted through manual endpoint selection. To analyze the Vessels and calculate the VTI, we used the method introduced by Khansari et al. . VTI calOur results showed that male patients with classic FD had the highest mean, median, and individual data points of VTI compared to all other subgroups. As male patients with classic FD usually have a more severe progression of the disease compared to other subgroups of patients with FD, retinal vasculature can be more altered in these patients.VT in patients with FD is speculated to be a result of intracytoplasmic storage of Gb3 within the endothelial cells, which can lead to endothelial proliferation and weakening of the vessel walls ,13,32. HStudies have shown that further changes in retinal vasculature and structure were found in patients with FD, e.g., an enlarged foveal avascular zone (FAZ) or increased vessel density (VD) and vessel length density (VLD) in the SCP and deep capillary plexus (DCP) in patients with FD compared to HC, but also hyperreflective foci in OCT images of patients with FD as a sign of accumulation of Gb3 in the retina ,14. In aAs stated in previous studies, Gb3 accumulation plays an essential role in the pathogenesis of FD and the severity of clinical manifestation in patients with FD can vary enormously depending on the activity of GAL and thereby the accumulation of Gb3 in several tissues’ lysosomes and fluids ,5,6,7.Retinal VT has been described in many systemic diseases and genetic disorders, such as systemic hypertension, diabetes mellitus, chronic anemia, and retinopathy of prematurity . RegardiThere are several approaches to calculating a VTI, whereas the main approaches can be divided into a distance approach, a curvature approach, or a mixed approach. The distance approach calculates the tortuosity as a quotient of the length of a vessel (arc length) over a straight line (chord length) between the two endpoints or branching points of the measured vessel or vessel section respectively.For the curvature approach, an index is calculated that provides the minimum of changes in direction of the blood flow within a tortuous vessel. This approach has also several alternative ways of being calculated, as described by Abdalla et al. . A remarThe mixed approaches are methods that combine both previously described approaches, such as the one proposed by Khansari et al. . This apWhile being a large cohort of patients for a rare disease such as FD that received OCTA imaging, the number of cases remains low, especially in our subgroup analysis. In addition, we had to work with a non-age-matched cohort for comparison, limiting the comparability between the two cohorts. A further limitation is this study’s retrospective nature and the initially stated situation of various approaches in the literature regarding the assessment of retinal VT, making it difficult to compare our findings to other examples from the literature. Yet, we decided to take the advanced approach of a mixed model, as outlined above, to calculate the mean VTI per 6 × 6 mm OCTA image of all vessels in the SCP. However, we only assessed structural changes of retinal vessels with this approach, whereas no statement can be made regarding the dynamics of the retinal blood flow or further structural changes in the retinas of patients with FD. Nevertheless, OCTA has proven a reliable noninvasive imaging modality for determining microvascular changes in the retina, being a modality for screening and follow-up examinations in patients with retinal vascular diseases, including patients with common systemic diseases, but also patients with rare systemic diseases such as FD ,58,59,60We could detect a significantly higher retinal VT in male patients with classic FD compared to all other subgroups in our study. Further investigations of retinal VTI in patients with FD could strengthen the role of OCTA as a noninvasive screening and follow-up imaging modality to assess disease severity and progression in affected patients.


This study employs the phase-field regularized cohesion model (PF-CZM) to simulate crack propagation and damage behavior in porous granite. The impact of the pore radius (r), initial crack–pore distance (D), and pore–crack angle (θ) on crack propagation is investigated. The simulation findings reveal that, with a fixed deflection angle and initial crack–pore distance, larger pores are more likely to induce crack extension under identical loading conditions. Moreover, with r and θ remaining constant, the crack extension can be divided into two stages: from its initiation to the lower edge of the pore and then from the lower edge to the upper boundary of the model. Multiple combinations of different D/r ratios and pore radii are derived by varying the values of D and r. These results demonstrate that with a constant r, cracks tend to deflect towards the pore closer to the initial crack. Conversely, when D remains constant, cracks will preferentially deflect toward pores with a larger r. In summary, the numerical simulation of rock pores and initial cracks, based on the PF-CZM, exhibits remarkable predictive capabilities and holds significant potential in advancing rock fracture analyses. The pore structure within rock masses encompasses heterogeneous elements such as voids, pores, and cracks . NumerouNumerous experiments and numerical simulations have been deployed to scrutinize the intricate behavior of rocks containing defects, encompassing aspects such as crack initiation, propagation, interconnection, and linkage. The numerical manifold method was proposed to analyze the initiation and propagation of frictional cracks in rocks . The faiRecently, the phase-field model (PFM) ,15,16 haWong et al. ,36 demonIn rock fracture research, studies on rock fractures are mainly focused on single-pore media, and there is a lack of systematic research on the fracture behavior of porous media. In this work, a novel unified phase-field regularized cohesive force model was proposed to study the expansion of porous media in rock, especially for double-pore structures. This model allowed us to describe the fracture behavior in rocks in a continuous way, avoiding the discretization problem in the traditional methods and improving the numerical stability and convergence of the model. In this study, the accuracy of the model was validated through experimental verification and numerical simulation. Two classical rock fracture cases were simulated and analyzed to demonstrate the effectiveness and accuracy of the phase-field regularized cohesive model in predicting rock fracture behavior.n. The deformation of the solid primarily results from both body forces In this section, we provide a concise overview of the phase-field theory of damage and fracture . Figure The sharp crack The phase-field fracture theory is underpinned by the coupled damage–displacement problem . These gThe term ge model ,41.This paper focuses solely on the cracking behavior under monotonic loading conditions, with the simplest constitutive relations  shown asTo maintain generality, the general expression satisfying condition (4) is as follows :(7){α(d)With the parameters  studies  have empSeveral popular brittle fracture phase-field models ,43,44 arFor the above brittle fracture phase-field models, the calculation equation for the critical (peak) stress given in ,44:(10)σThe critical stress ameter b . To addrarameter . A prevaIn brittle fracture phase-field models, the internal length Comparatively, for any parameter strength :(12)σc=2In other words, parameter th scale ,44,47. C.’s work  illustraIn this paper, we refer to Wu et al.’s research  for the The ratio of Based on the linear softening curve, it can be deduced that The resulting PF-CZM is considered optimal since it accommodates a wide range of softening laws commonly employed for brittle and quasi-brittle materials, including linear equations, exponential equations, hyperbolic equations, and the approach proposed by Sarac et al. . BrittleThe mentioned parameters shown in .For the PF-CZM, i.e., Once the crack nucleates, i.e., Or, equivalently,Therefore, as the maximum principal stress In this section, we employed the PF-CZM for numerical simulation and experimental verification on rocks. We conducted two sets of experiments, namely, the SCB monotonic fracture test  and the A typical cement-stabilized sand was characterized with the Semi-Circular Bend (SCB) fatigue test for crack propagation . The sanRezanezhad et al.  investigeference . Figure eference , and theIn this section, we utilized the PF-CZM to numerically simulate the relative location of rock pores and initial cracks. Pores and cracks are typical defect patterns found in rocks. This model enabled us to analyze phenomena such as crack propagation and rock deformation in the rock, thus enhancing our understanding of the mechanisms governing rock failure by simulating the crack evolution under varying conditions. The relative location of pores and initial cracks are shown in The initial crack length (a), rock slab length (L), and rock slab width (W) were 1, 15, and 10 mm, respectively. u is denoted as the displacement load. Our investigation primarily focused on three key aspects: (1) We explored the impact of varying pore sizes (r) on crack propagation. This variation allowed us to observe how pore size influenced rock fracture and strength. (2) Considering the variation of different pore-to-crack distances (D) on crack propagation, we gained insights into the varying D interaction effects on crack propagation by adjusting the value of D. (3) The pore–crack angle (θ) describes the deflection of the pore position relative to the crack tip. Varying the value of θ enabled us to assess the impact of the relative position of pores in the rock on the crack path propagation and velocity.To investigate the influence of varying r on crack propagation, we kept certain parameters constant: D = 4 mm, θ = 90°, and a = 1 mm. We explored different pore sizes, specifically r values of 0.2, 0.8, and 1.2 mm. A uniform displacement of 0.001 mm/s was applied to both the left and right sides of the model, simulating the crack propagation process, which allowed us to determine the influence of different pores on rock failure.The fracture diagrams for crack propagation with different pore sizes are illustrated in From the load–displacement curves shown in In this section, we delved into the impact of the pore-to-crack distance (D) on crack propagation. Referring to the schematic diagram of the relative positions of pores and cracks in Throughout the process, from the crack initiation to its expansion towards the lower edge of the pore, the reaction force steadily increased. In this first stage, the maximum reaction force reached 440, 472, and 482 N for Ds of 1, 1.5, and 2 mm, respectively, and slightly rose by 7% and 9%. As the crack continued to expand from the lower edge to the upper edge, higher loads were applied compared with those of the previous stage. In the second stage, the maximum reaction force was 566, 543, and 522 N for Ds of 1, 1.5, and 2 mm, respectively, and slightly decreased by 4% and 7%. These results indicated that a higher load was required to resist crack propagation as the crack extended from the bottom edge to the top edge. This conclusion was consistent with Zhang et al.’s research [In the previous section, we explored various scenarios where the θ was 90°. In such cases, we observed that the typical pattern of crack growth involved a straight-line extension throughout the pore, reaching the upper edge. Meanwhile, the stress distribution in the crack tip region was usually symmetrical. However, when the angle between the pore and the crack was not 90°, crack propagation often deflected from a straight path toward the direction of pores at a specific angle, and the stress distribution in the crack tip region was no longer symmetrical. It should be noted that not all pores exhibit this effect, and only pores located at critical angles related to the crack tip can cause such crack deflection.To visually illustrate the positional relationship between the angle θ of double pores and fractures, we created a simplified model, as depicted in Building upon the findings from To simulate the influence of different deflection angles on crack propagation, we employed four models, namely, Model *2, Model *3, Model *5, and Model *8, as listed in Continuing the simulations for Models *5 and *8, which, as shown in Based on the previous simulation results, we could draw the following conclusions. When r remained constant and D varied, the crack deflected towards the closer pore, indicating that D was the most influential parameter affecting crack deflection. Alterations in D gave rise to changes in stress distribution, which, in turn, affected the crack propagation rate between the regions of high and low stress and then determined the crack deflection direction.In The second type of simulation adopted Models *6 and *7 (θ = 55°), in which the radii were 1.2 and 0.4 mm, respectively, and the ratios of D/r were three and four, respectively, and Models *9 and *10 (θ = 65°), in which the radii were 1.2 and 0.4 mm, respectively, and the ratios of D/r were three and four, respectively. With the crack propagation and von Mises stress distribution shown in In order to verify the correctness of this principle, Models *11 and *15 were simulated in This study employed the PF-CZM to verify the SCB disk fracture test, revealing a strong concurrence between our simulations and experimental results. Additionally, we applied this model to analyze porous granite and compared the simulation results with previously studied experimental results, which demonstrated an extremely high agreement between these results. This underscored the remarkable effectiveness of the PF-CZM as a numerical tool for precisely forecasting porous rock crack propagation. This study offered a dependable numerical tool, enhancing our comprehension of porous rock characteristics and damage across diverse conditions.To investigate the effects of pores and initial cracks on crack propagation, three different simulations were carried out.In a tensile model with an initial crack and two pores ), the crack would deflect towards the pore with the smaller pore distance.In a tensile model with an initial crack and two pores  but different pore radii), the crack would deflect towards the pore with larger pore radii.In a tensile model with an initial crack and two pores (with different crack–pore distances (Ds) and pore radii), the crack would deflect towards the pore with the smaller D/r ratio.When rock materials are applied in complex environments such as deep seas and high temperatures, they will face challenges such as chemical corrosion and thermal strain, which can cause irreversible damage to rock structures. In this context, it is necessary to consider the effects of mechanical–chemical coupling and thermos–mechanical coupling. By establishing the mathematical model of mechanical, chemical, and temperature coupling, the crack propagation behavior of rock materials can be accurately simulated under harsh environment.