The Summer School on Pharmaceutical Analysis (SSPA) addresses the most advanced analytical methodologies involved in the development of new drugs and in drug manufacturing. The ultimate goal of a drug discovery program is to generate a candidate drug compound that, besides being active and selective, is safe and efficacious in humans. In order to develop such a compound, many criteria including affinity, activity, selectivity, physico-chemical properties, and pharmacokinetics must be optimized. The definition of these criteria involves the availability and knowledge of appropriate analytical methodologies.
Each SSPA edition is focused on a specific aspect/phase of the drug discovery process. In detail, the 2024 edition of SSPA will focus on New therapeutic approaches, new analytical challanges.
Lectures will be delivered by recognized experts from the academic and industrial sectors. A list of confirmed lecturers is available at the section “meet our speakers”. The School social activities are planned to promote networking and favor interactivity between participants and lecturers. To maximize these aspects the number of participants in presence is limited to 80.
We are working to make SSPA2024 a great opportunity for enforcing your scientific competitiveness but also an enjoyable and friendly experience.
We are looking forward to welcoming you in Rimini.
Chair of SSPA
Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna
18th-20th Sept 2024
14 Drug Discovery Professionals
The ultimate goal of a drug discovery program is to generate a candidate drug compound that, besides being active and selective in vitro on the isolated target, is safe and efficacious in humans. In order to develop such a compound, many criteria including affinity, activity, selectivity, physico-chemical properties, and pharmacokinetics must be optimized. The definition of these criteria involves the availability and knowledge of appropriate bio-analytical methodologies.
The SSPA is housed in Aula 7 of the Alberti Complex (Aula Alberti 7), via Carlo Cattaneo 17, Rimini. Venue location is mapped here below. The school venue is conveniently located at a walking distance from the rail station and in Rimini city center.
September 14th, 2023
SSPA2023 is now over
Thanks to all participants and lecturers to have made this edition of the School so special. See you next year!
Human serum albumin (HA), the most abundant protein in plasma, plays a vital role in maintaining physiological homeostasis and holds great clinical significance.  While its primary function as an oncotic agent is stabilizing intravascular volumes, HA exhibits a wide range of non-oncotic properties. These include scavenging free radicals and binding and transportation of endogenous and exogenous molecules. Notably, HA's binding of drugs at specific sites significantly influences the pharmacokinetics and pharmacodynamics of many commonly prescribed medications. 
However, various structural alterations can occur during HA plasma lifespan, including truncation at the N and C terminals, glycation at lysine and arginine residues, and reversible and irreversible oxidization at the Cys34 residue - the only cysteine residue not involved in intramolecular disulfide bridges. These modifications have garnered scientific interest as they may serve as potential markers for certain pathological conditions. In chronic diseases like kidney failure, cirrhosis, and diabetes mellitus (DM), inflammatory and oxidative processes can lead to an accumulation of modified forms of HA. ,  In type 2 DM (T2DM), one of the most common complications is diabetic kidney disease (DKD). Currently, common clinical practice for assessing DKD severity involves measuring estimated glomerular filtration rate (eGFR) and albuminuria. However, not all patients with abnormal albuminuria or impaired renal function progress to end-stage renal disease (ESRD).  For this reason, intense research aims at finding additive markers and developing new methods for an early detection of renal dysfunction.  According to a recent study, which focuses on HA structural alterations in patient affected by decompensated cirrhosis, the relative abundance of native HA (nHA) decreases at a higher extent than the total HA concentration (tHA). From this remark, Caraceni and coworkers have introduced the concept of effective albumin concentration (eHA), namely the concentration of albumin in its native form. In the frame of liver cirrhosis, this new parameter showed to be more closely associated with disease severity, carrying a greater diagnostic power. 
To delve deeper into understanding the structural and functional aspects of HA alterations, part of my PhD project was devoted to the development of analytical methods to characterize HA structural alterations using LC-MS and to assess whether nHA, tHA and eHA possibly correlate HA dysfunction with the severity of renal damage in DKD patients. Additionally, Surface Plasmon Resonance (SPR) technology was employed to investigate HA functionality, particularly its binding capacity. To address this, a sensor chip able to selectively capture the patient's pool of circulating forms of HA was developed.
The project was pursued in collaboration with the Unit of Metabolic Diseases & Clinical Dietetics and the Unit of Nephrology, Dialysis and Transplantation at the S. Orsola-Malpighi Hospital in Bologna, which oversaw patient recruitment. Patient recruitment was started within an intramural project financed by the University of Bologna (AlmaIdea). The subject population consisted of 117 diabetic patients suffering from T2DM, diagnosed for at least one year and classified upon albuminuria and eGFR parameters at different stages of renal impairment (T2DM+DKD from I to III). To determine tHA, nHA and eHA for each patient, a colorimetric assay and a previously developed LC-MS method were exploited, respectively for the quantification of HA in plasma samples and for the characterization and determination of the relative abundance of all HA forms in the diabetic population under investigation. tHA was, indeed, determined by bromocresol green (BCG) colorimetric assay. High-performance liquid chromatography coupled to electrospray ionization/quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-ToF) was used to assess the relative amount of nHA and its microheterogeneity profile. LC-MS analysis showed that HA structure was significantly altered in the T2DM+DKD group. In particular, a significant increase in the cysteinylated form (termed human non-mercaptoalbumin 1, HNA1) was observed. Consequently, a significant decrease in the relative abundance of both nHA and human mercaptoalbumin (HMA), in which the Cys34 thiol group is not oxidized, was also encountered. The small but significant decrease in irreversibly oxidized HA forms at Cys34 (commonly named as non-mercaptoalbumin 2, HNA2) in the T2DM+DKD patients was not entirely unexpected. Indeed, a similar result was previously reported in a similar study performed on cirrhotic patients.  In that study slightly lower HNA2 levels were found in inpatients than outpatients. Finally, the glycated form of the protein increased slightly, whereas the abundance of the truncated forms did not differ significantly. The analysis of the three parameters under study revealed that T2DM+DKD patients exhibited a slightly lower amount of tHA compared to T2DM-DKD patients, consistent with previous studies in which reduced HA levels were linked to increased inflammation, reduced hepatic synthesis, catabolism, and vascular permeability in disease states associated with heightened inflammatory processes like DKD. In addition to serum HA concentration, the structural integrity of HA was found to be more compromised in patients with renal impairment; as evidence, nHA in T2DM+DKD patients is significantly lower than in T2DM-DKD patients. This finding is in line with recent research that has shown that oxidative changes in HA parallel the disease-related increase in oxidative stress. Notably, nHA and eHA proved to be more informative than tHA, as those parameters consider the protein's structure alongside its serum concentration. tHA, nHA and eHA were also evaluated for possible correlation with the clinical parameters that best describe the severity of DKD and are currently used in clinical practice: (i) creatinine, (ii) microalbuminuria and (iii) eGFR. As expected, a negative correlation was observed between the three forms of HA, creatinine and albuminuria levels, whereas a positive correlation was observed in the case of eGFR. This means that the presence or absence of kidney damage affects both structural integrity and the level of albuminuria. Again, nHA resulted to be more informative than tHA (higher correlation coefficients), indicating that renal impairment affects HA structure rather than the amount of circulating HA. More interestingly, the ROC curve analysis showed that the parameters endowed with the better diagnostic power toward renal impairment are nHA and eHA.
Several studies have been performed by several groups to assess whether HA binding capacity is affected by structural modifications in specific pathological conditions. [9-11] Results from those studies are not all convergent and, when carried out in vitro under stress conditions, often refer to highly altered species and to a single specific alteration such as glycation. Conversely, the natural microetherogenity of circulating HSA implies a series of alterations, including reversible and irreversible oxidation, glycation and truncation, simultaneously occurs, as also evidenced by MS-based studies. This highlights the inherent complexity and diversity of HA modifications in physiological and pathological conditions. In this scenario, SPR technology was exploited to evaluate whether structural alterations occurring in circulating HA in patients could impact its binding properties. It is worth mentioning that drug binding to HA can impact drug properties such as plasma solubility and ADME (absorption, distribution, metabolism, and excretion) profile. Since alterations occurring on HA snapshot patient-specific conditions and are influenced by multiple factors, it may be relevant, in the light of a more personalized medicine, to investigate patient-specific HA structural alterations and their impact on HA binding properties. To achieve these objectives, a commercially available sensor chip coated with a carboxy-methyldestrane layer on a gold surface (chip CM5, Cytiva®) was utilized, and an anti-HSA antibody was immobilized on the chip surface through a well-established covalent binding procedure (amine coupling). Chip functionalization, reversible HA capture, surface regeneration and sample handling were evaluated and optimized. For sensor chip validation, three different well-known and commercially available markers were screened: phenylbutazone (PBZ), dansyl-phenylalanine (DAP) and biliverdin (BVD), respectively a site I, a site II and a site III marker. For each of them, the steady-state dissociation constant (KD) was assessed and compared with binding data available from the literature. Furthermore, the affinity of teicoplanin (TEICO), a non-site-specific HSA binder, was also evaluated.
While it is now known that structural damage to HA occurs in patients with cirrhosis, it remains unclear whether these alterations also affect its biological functions and drug binding. To elucidate these aspects, the validated sensing surface was used to perform a preliminary ex vivo investigation of HSA binding functions in patients suffering from decompensated cirrhosis. As a pilot study, plasma samples from six patients admitted to with acute-on-chronic liver failure (ACLF) at the S. Orsola-Malpighi Hospital in Bologna and six age-matched healthy control subjects were selected. Results showed that the binding affinity of PBZ to site I is slightly higher in cirrhotic patients than in healthy control subjects. In contrast, sites II and III showed no significant differences in affinity towards DPA and BDV. At the level of these sites, it can be assumed that the structural changes that HSA undergoes in cirrhotic patients do not significantly affect its binding capacity at the three high affinity binding sites. A different behavior was observed for TEICO for which a significant reduction in its binding affinity (higher KD value) was encountered. As TEICO is an antibiotic of choice in the treatment of infections in cirrhotic patients, this result is of potential clinical relevance and requires further evaluation.
Following these findings in cirrhotic patients with liver failure, TEICO was chosen as a probe to preliminary assess whether albumin binding capacity was also altered in patients with T2DM with renal impairment. As a pilot study, 20 diabetic patients were selected in order of enrolment: 10 without renal damage (T2DM-DKD) and 10 with severe renal damage (T2DM+DKD III), classified based on eGFR and albuminuria levels. Despite the significant HSA structural damage found in this population, as determined by LC-MS, the binding capacity of TEICO was not significantly affected.
This research emphasizes the importance of investigating both HSA structure and functionality of HA since correlation of them may offer a better understanding on the impact of pathological conditions on HA function. The developed patient-specific sensing surfaces offer promising opportunities for patient-specific investigations also in the light of a more personalized therapeutical intervention, paving the way for further investigations into HA alterations in various pathologies characterized by oxidative stress and inflammation.
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Naturally or chemically derived complex drugs are products with highly heterogeneous molecular components. The population of components present in the mixtures is variable and the active part is often not distinguishable within the product. Heparin, an anticoagulant drug used to prevent blood clots in surgery and to treat various disorders in which there is an increased risk of blood clot formation, is perhaps one of the best examples of complex drug. Heparin is extracted from porcine and more rarely bovine intestine mucosa. The process starts at the animal slaughtering, where the mucosa is separated from casing and heparin isolated, together other glycosaminoglycans, to obtain the so called crude heparin. While this part of the production takes place in not completely regulated environments, the final steps of purification leading to the API are performed in GMP complying plants . Before 2008, the complexity of heparin supply chain and the lack of specific tests for controlling the quality and purity of heparin products had failed to prevent contaminated batches to enter in the market, resulted in 94 deaths and about 600 adverse reactions [2,3].
The heparin contamination crisis has highlighted the need of implementing a panel of new analytical methods able to elucidate the molecular properties of heparin. Particularly, proton NMR tests were introduced in the monographs of several pharmacopoeias for the purpose of identification and characterization of the active principle . However, only the combined used of different techniques, including 2D-NMR, LC-MS, molecular weight determination, often coupled with statistical analysis of spectra of sample libraries, can guarantee the quality of the product from intentional contamination or from process deviation. Moreover, the development and validation of these methods have made it possible to introduce useful analytical tools for regulatory agencies to identify the process signatures necessary to demonstrate the equivalence of generic versions of low molecular weight heparins to their branded products . The same approaches are currently applied to other complex drugs of polysaccharidic, polypeptidic and polynucleotidic nature .
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Even though Cannabis sativa L. is among the plants known for the longest time for its pharmacological properties, studies regarding the composition of active ingredients have started only in the last decades. Cannabis sativa L. is a prolific producer of a peculiar group of isoprenylated resorcinyl polyketides well known as phytocannabinoids. Over 150 phytocannabinoids are produced in the plant of which only few have been isolated and characterized. In the early '60s, Prof. Raphael Mechoulam identified the two main phytocannabinoids, the psychotropic &DELTA;9-tetrahydrocannabinol (&DELTA;9-THC) responsible for the euphoriant effects of cannabis and the non-psychotropic cannabidiol (CBD). Numerous scientific works and clinical trials have described their biological activity. All phytocannabinoids produced in the plant are in carboxylated form (e.g. &DELTA;9-tetrahydrocannabinolic acid (&DELTA;9-THCA) and cannabidiolic acid (CBDA)). Over time and under specific climatic conditions they undergo a spontaneous decarboxylation process to give the corresponding decarboxylated phytocannabinoids such as &DELTA;9-THC and CBD. To date, cannabis is a plant with countless applications, including the treatment of muscle and neuropathic pain, Tourette's syndrome, etc. [2,3] However, very little is known about the complex chemical composition of cannabis, which differs according to variety, climatic conditions of growth, cultivation techniques and the presence of biotic and/or abiotic stresses. The phytocannabinoid composition affects the pharmacological profile of cannabis extracts. From this point of view, the stereochemistry of phytocannabinoids also plays an important role. For example, (+)- and (-)- isomers of trans-CBD proved to have contrasting biological activities. In detail, unlike (-)-trans-CBD, which has no affinity for either CB1 or CB2 receptors, (+)-trans-CBD shows a stronger binding affinity for CB2 and enhanced affinity for CB1 receptors, though not as high as (-)-trans-&DELTA;9-THC. [4,5] Given that, the evaluation of the stereoisomeric composition of the phytocannabinoids is extremally important, especially in medicinal cannabis extracts.
In light of the above, the aim of my PhD program is to identify new phytocannabinoids and assess the stereoisomeric composition of phytocannabinoids to give a complete overview of the chemical composition of Cannabis sativa extracts. This is combined with the evaluation of their pharmacological activity. Moreover, the project aims to develop sensitive and selective analytical methods to quantify such phytocannabinoids.
With this purpose, in the first part of my PhD project, an analytical method based on high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) was developed in an untargeted metabolomics fashion. This allowed us to putatively identify numerous carboxylated and decarboxylated phytocannabinoids present in a complex biological mixture: Cannabidihexol (CBDH) with antinociceptive activity in mice, Tetrahydrocannabihexol (&DELTA;9-THCH), Cannabigerobutol (CBGB), Cis-&DELTA;9-tetrahydrocannabinolic acid (cis- &DELTA;9-THCA), &DELTA;9-tetrahydrocannabiphorolic acid (&DELTA;9-THCPA), Cannabidiphorolic acid (CBDPA).[6-9] To confirm their identity they were isolated, fully characterized and their chemical properties compared to those of the corresponding synthetic species. To this end, ad hoc synthetic strategies were developed and the pure synthetic compounds were used as reference standards for the development of sensitive and selective HPLC-UV-HRMS methods under a targeted metabolomics fashion in order to quantify the new phytocannabinoids in different cannabis varieties.
In the second part of my PhD project, we focused our attention on the stereochemistry of the two phytocannabinoids approved for some therapeutic indications: &DELTA;9-THC and CBD.[10,11] These compounds possess two chiral centers responsible for the potential existence of four stereoisomers: (-)-trans-, (+)-trans-, (-)-cis-, and (+)-cis-. To date, we know that nature prefers (-)-trans isomers. Moreover, cis isomers of CBD and (+)-trans-CBD have never been found in cannabis, while the cis isomers of &DELTA;9-THC and traces of the (+)-trans isomer of THC have been recently found in fiber-type cannabis plants and in the medicinal cannabis variety Bedrocan (Bedrocan B.V., The Netherlands), respectively.[12,13] However, no other studies have been carried out to confirm the presence of this minor stereoisomer in other varieties and no analytical method has been published on the chiral separation of the carboxylated phytocannabinoid, which instead gives the real picture of what the plant produces enzymatically.
Whit this purpose, chiral HPLC methods coupled to a diode array detector (DAD) and to high-resolution mass spectrometry (HRMS) were developed and optimized. The analytical standards, (+)-trans- Δ9-THC, (-)-trans- &DELTA;9-THC, (+)-trans-CBD, (-)-trans-CBD and their carboxylated counterparts ((+)-trans- &DELTA;9-THCA, (-)-trans- &DELTA;9-THCA, (+)-trans-CBDA, and (-)-trans-CBDA) were not commercially available, therefore a stereoselective synthesis was performed.
In order to assess the presence of (+)-trans isomers of THCA, CBDA and corresponding decarboxylated forms in the Italian medicinal cannabis variety FM2, the two phytocannabinoids trans-THCA and trans-CBDA were isolated, each in an individual fraction, by achiral reversed phase (RP) HPLC-DAD-HRMS (Poroshell 120 EC-C18, linear gradient from 60% to 95% ACN (with 0.1% formic acid) in 15 min, followed by isocratic elution at 95% ACN, washing step at 98% ACN for 3 min and re-equilibration with the initial conditions, total run time 26 min, flow rate 0.5 mL/min). After decarboxylation of the starting plant material at 130 ºC for 2 hours, the corresponding decarboxylated species trans-CBD and trans-&DELTA;9-THC were isolated on the achiral dimension. With the isolated compounds in hand, a chiral analysis was carried out by HPLC-DAD-HRMS with the column CHIRALPAK AD-RH (150 x 4.6 mm I.D., 5 μm) with the following settings:
- The enantiomers of trans-CBD and trans-&DELTA;9-THC were separated with 60% ACN setting the column compartment temperature at 30 ºC and the flow rate at 1.5 mL/min.
- The enantiomers of trans-CBDA and trans-&DELTA;9-THCA were separated with 50% and 75% 2-propanol, respectively. Both at 30 ºC and 1 mL/min. At the limit of the sensitivity of the developed methods the main phytocannabinoids, trans-CBDA, trans-&DELTA;9-THCA, trans-CBD and trans-&DELTA;9-THC were found as single (-)-enantiomers in the Italian medicinal cannabis variety FM2. In the conditions applied the decarboxylation process did not affect the original stereochemistry of the species under investigation.
The development of an achiral-chiral chromatographic method proved to be very useful in the assessment of the stereoisomeric composition of the main phytocannabinoids in a cannabis extract. In a broader context, the designed work provides the great advantage to disclose the presence of enantiomeric impurities of single isolated peaks without heating the starting material, which would otherwise lead to an altered phytocannabinoid composition.
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The enantioselective analysis of illicit substances in different matrices (including biological and environmental samples as well as samples from seizures) is of prior importance since it allows to obtain relevant information concerning, inter alia, (i) the distinction between their legal and illicit use; (ii) the distinction between their direct consumption or their presence as a result of the metabolic fate of other compounds; (iii) the identification of the synthesis protocols applied in clandestine laboratories, along with the chemical identity of the employed precursors; (iv) the monitoring of changing patterns of drugs abuse; (v) the differentiation between their direct consumption and the deliberate disposal of unused material in the sewage; and (vi) the estimation of the actual environmental risk [1-8].
Going into more detail, as far as the first two points in the above list are concerned [points (i) and (ii)], the case of enantiomeric composition of amphetamine and methamphetamine is highly explanatory [1,2,8]. Indeed, several therapeutic medicines contain, or are metabolized to, amphetamine and methamphetamine enantiomers and are excreted in urine. For example, in both the USA and in some European Countries, racemic and (S)-amphetamine, as well as (R)- and (S)-methamphetamine, are prescription medications, and moreover other compounds [such as (R)-selegiline and famprofazone, just to cite but a few] have been shown to be precursors of amphetamine and/or methamphetamine as a result of their metabolism. In this framework, taking also advantage of the exact knowledge about the stereoselective metabolic pathway of these two compounds, their enantiomeric composition in biological and environmental samples can contribute to distinguish between their medical (licit) or illicit use [1,2,8]. Going forward in the list [points (iii) and (iv)], it is very well known that the methods used for the illegal synthesis of many psychostimulants may differ both in their precursors and in the enantiomeric composition of the resulting products, and are subject to dynamic change due to limitations of the selected precursor(s) [3,4]. Due to either local or international restrictions on precursor chemicals, clandestine laboratories are indeed ever more frequently forced to switch to alternative starting materials and different pathways of synthesis (both enantioselective and non-enantioselective). This "technology transfer" allows clandestine producers to escape from applicable laws, whereas it leaves specific "stereochemical fingerprints" in the produced substances, which can be unveiled through the application of enantioselective analysis protocols (thereby allowing the possibility to control the legal market of some precursors) [5,6]. Indeed, differences in enantiomer ratios imply that the compounds may have been synthesized from different precursors by different methods, eventually in different Countries. The last two points in the list [points (v) and (vi)] enter the frame of the so-called "sewage epidemiology" also known as "wastewater-based epidemiology" [1,2,4,5]. This approach is based on the idea that screening for drugs which are consumed by subjects, eliminated through biological fluids and reach wastewater sewage treatment facilities may be one of the fastest, most accurate ways to assess a community's drug use. Illicit drugs (as well as many drugs for therapeutic uses) enter the aquatic environment mainly through treated (or raw) sewage mostly from domestic households as a result of their use. They can be present in the environment in the form of parent unaltered compounds or metabolites, or can undergo additional transformation during wastewater treatment to produce compounds of possible concern to humans and wildlife [1,2,4,5]. According to this "sewage forensics" approach, the capacity to define the enantiomeric composition of chiral illicit drugs in the environment is of great importance as it allows to distinguish illicit drug abuse from direct disposal of unused material. Moreover, since the activity and toxicity of these compounds are often dependent on stereochemistry, it is of fundamental importance to understand the influence of wastewater treatment processes in the (stereo)selective degradation of chiral drugs in order to improve the performance of wastewater treatment plants (WWTPs) and to protect the receiving aquatic environment [1,2,4,5].
Many of the investigation related to the above points have been mostly addressed to the group of amphetamines, while little is still known about other classes of illicit drugs, especially as far as the so-called novel psychoactive substances (NPSs) are concerned. This justifies the necessity to develop methods enabling the easily accessible and accurate enantioselective analysis of other classes of psychoactive substances than amphetamines in different matrices [7,8].
During my PhD Research period, I have been deeply involved in the development of very efficient methods for the enantioselective LC and LC-MS analysis of fentanyl derivatives, benzofuran-substituted phenethylamines, substituted tryptamines, substituted cathinones, as well as synthetic cannabinoids, with a rich variety of chiral stationary phases (CSPs) incorporating polysaccharide- (cellulose and amylose type), crown-ether-, and Pirkle-type chiral selectors [9,10]. In these studies, all most relevant elution modes [that is, reversed- and normal-phase (RP, NP), as well as polar-organic or polar-ionic (PO, PI) conditions] for LC applications have been applied with mobile phases fully compatible with MS detectors. The applicability of the developed enantioselective LC methods to the study of real biological samples has been also appraised on MS-based platforms, through the coupling to miniaturized microsampling technologies. Also importantly, the combination of circular dichroism spectroscopy (ECD) to time-dependent density functional theory (TD-DFT) calculations has allowed the full stereochemical characterization of the analyzed enantiomers in absence of pure enantiomeric standards.
In the first study, the enantioseparation of four fentanyl derivatives, that is, (±)-trans-3-methyl norfentanyl, (±)-cis-3-methyl norfentanyl, β-hydroxyfentanyl, and β-hydroxythiofentanyl, was obtained under PI conditions. Indeed, the use of acetonitrile-based mobile phases with minor amounts of either 2-propanol or ethanol (plus diethylamine and formic acid as ionic additives) allowed obtaining enantioseparation and enantioresolution factors up to 1.83 and 7.02, respectively. For the study, the two chiral stationary phases (CSPs) cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(4-chloro-3-methylphenylcarbamate) were used, displaying a remarkably different performance towards the enantioseparation of (±)-cis-3-methyl norfentanyl. Chiral LC analyses with a high-resolution mass spectrometry detector were also carried out in order to confirm the obtained data and demonstrate the suitability and compatibility of the optimized mobile phases with mass spectrometric systems .
In the second study, the two Pirkle-type (R,R)- and (S,S)- Whelk O®1 CSPs based on enantiomeric 1-(3,5-dinitrobenzamido)-1,2,3,4,-tetrahydrophenanthrene chiral selectors were used for the first time for the enantioseparation of five synthetic cannabinoids. The enantioselective analysis of the indazole-based 5-chloro-AB-PINACA, AB-CHMINACA, 5-fluoro-APP-PINACA, AB-FUBINACA, APP-FUBINACA was succeeded in less than 30 min under RP conditions with water/acetonitrile-based eluents. The enantiomeric elution order (EEO) was apprised by coupling electronic ECD analysis to in silico TD-DFT calculations. The application of the "inverted chirality column approach (ICCA)", combined to the results of RP analysis with the Lux® i-Cellulose-5 carrying the immobilized cellulose tris(3,5-dichlorophenylcarbamate) chiral selector, allowed to confirm the EEO with the Whelk O®1 phases. The developed methods were successfully coupled to a novel dried microsampling approach based on microfluidic generated-dried blood spot (mfDBS) technology for the accurate collection of whole blood microvolumes. The proposed miniaturized strategy by means of microfluidic channel-based devices provide several advantages in terms of collection, storage, and handling compared to classical blood and plasma processing, and all the main parameters involved in mfDBS sampling, storage, handling, and pretreatment were optimized in order to obtain straightforward, yet effective and reliable protocols. Satisfactory validation results were obtained for the microsampling platform, in terms of mean extraction yields (> 86.8%), precision (RSD < 5.9%), and stability (< 5.5% analyte loss after 30 days). The original microsampling methodology has been successfully exploited for a proof-of-concept application to fortified whole blood miniaturized samples proving to be suitable for the enantioselective quali-quantitative analysis of indazole-based synthetic cannabinoids.
In the third and last study, efficient methods for the enantioselective HPLC analysis of five benzofuran-substituted phenethylamines, two substituted tryptamines, and three substituted cathinones were developed. For the first time, RP (eluents made up with acidic water-methanol solutions) and PI (eluent made up with an acetonitrile-methanol solution incorporating both an acidic and a basic additive) conditions fully compatible with MS detectors were applied with CSP incorporating the (+)-(18-crown-6)-tetracarboxylic acid chiral selector. Enantioresolution was achieved for nine compounds with a and RS factors up to 1.32 and 5.12, respectively. Circular dichroism (CD) detection, CD spectroscopy in stopped-flow mode and quantum mechanical (QM) calculations were successfully employed to investigate the absolute stereochemistry of mephedrone, methylone and butylone and allowed to establish a (R)<(S) EEO for these compounds on the chosen CSP. Whole blood miniaturised samples collected by means of volumetric absorptive microsampling (VAMS) technology and fortified with the target analytes were extracted following an optimised protocol and effectively analysed by means of an ultra-high performance liquid chromatography-MS system. By this way a proof-of-concept procedure was applied, demonstrating the suitability of the method for quali-quantitative enantioselective assessment of the selected psychoactive substances in advanced biological microsamples. VAMS microsamplers including a polypropylene handle topped with a small tip of a polymeric porous material were used and allowed to volumetrically collect small aliquots of whole blood (10 μL) independently from its density. Highly appreciable volumetric accuracy (bias, in the -8.7-8.1% range) and precision (% CV, in the 2.8-5.9% range) turned out .
 Evans S. E., Davies P., Lubben A., Kasprzyk-Hordern B., Anal. Chim. Acta 2015, 882: 112-126.
 Kasprzyk-Hordern B., Baker R. D., Sci. Total Environ. 2012, 423: 142-150.
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 Gelmi T.J., Verrijken M., Weinmann W., Regul. Toxicol. Pharmacol. 2020, 116: 104747.
 Wang T., Shen B., Shi Y., Xiang P., Yu Zh., Forensic Sci. Int. 2015, 246: 72-78.
 Lee J., Yang W. K., Han E. Y., Lee S. Y., Park Y. H., Lim M. A., Chung H. S., Park J. H., Forensic Sci. Int. 2007, 173: 68-72.
 Tedesco D., Di Pietra A. M., Rossi F., Garagnani M., Del Borrello E., Bertucci C., Andrisano V., J. Pharm. Biomed. Anal. 2013, 81: 76-79.
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 Varfaj I., Protti M., Cirrincione M., Carotti A., Sardella R., J. Chromatogr. A 2021, 1643: 462088.
 Protti M., Varfaj I., Carotti A., Tedesco D., Bartolini M., Favilli A., Sandro Gerli, Mercolini L., Sardella R., Talanta 2023, 257: 124332.
The analytical aspects related to development and manufacturing of an active ingredient are strictly connected to the quality of the drug substance.
The goal for the analytical development efforts is to ensure an appropriate support of process development activities with suitable analytical methods and specifications. The control of the impurities, their origin and fate to obtain an expected quality is one of the most important points of an API development. It is a regulatory requirement that all methods ensure conformity to acceptable scientific standards. The identification of the impurities using various analytical techniques requires a strong collaboration between Chemists and Analytical scientists.
A systematic approach has to be followed to find out the more suitable analytical controls with a proper level of precision and accuracy.
The solid-state characteristics of an Active Pharmaceutical Ingredient (API) have great impact on the fundamental properties of a medicinal product: solubility, dissolution rate, bioavailability, activity and toxicity, stability (shelf life identification) and manufacturing among others.
The knowledge acquired with the solid-state investigation of drug substance, drug products, and even intermediate of the manufacturing production helps to control and optimize the product performance and the drug delivery.
In the context of the QbD this became even more important because solid-state chemistry plays a critical role in many of the steps required to achieve QbD. In this system, the product is designed to meet patient requirements, the process is designed to meet product quality attributes, critical sources of process variability are identified and controlled and the process is continually monitored to ensure consistent quality over time. Moreover, the definition of the CQAs of the API (e.g. crystal form, particle size distribution, crystal morphology, etc.) influences the drug product attributes both in term of manufacturability and final bioavailability: the solid-state knowledge acts as a bridge between the DS and DP.
This presentation will introduce the Solid State / Physical Properties analytical techniques as well as examples on how the solid-state knowledge can influence a pharmaceutical product development through all the clinical phases to the market lunch and beyond.
Characterization is a critical aspect of Chemistry, Manufacturing, and Controls (CMC) development, which ensures that the drug product meets the highest standards for safety, efficacy, and quality. Most of the tests required for Drug product (DP) are the same present for Drug substance (DS) characterization. This presentation will focus on the differences when transitioning a method from DS to DP, how important sample purification and extraction steps are, what are the key aspects to be considered when working with Drug product. Besides the overview of additional testing necessary for Drug product, this presentation will focus on how analytical testing can support Drug Product development with a case of Dissolution testing. A dissolution procedure, from a quality control scope, should be robust, reproducible, and discriminatory, and should be able to assure a consistent product quality and detect product quality attributes, which, if altered, may affect in vivo performance. From an exploratory scope such as Bioavailability prediction, In vivo / In vitro correlation (IVIVC), Biorelevant in vitro dissolution testing can be used while discriminating dissolution testing have a crucial role during the initial formation development to select the most promising prototypes. An overview of Drug delivery systems and functionality testing which enable the targeted delivery or controlled release of a pharmaceutical drug into the body will be finally presented.
Drug administration by inhalation route is a way of delivering drugs that allows directly achieving the therapeutic site for obtaining a local or systemic effect; indeed, the inhalation therapy was initially focused on local diseases like asthma and COPD.
Contrary to other administration routes, the inhalation one requires a pharmaceutical product, which include both drug formulation and an inhaler device in order to afford the correct deposition of the drug in the respiratory tract.
The range of Orally Inhaled Drug Products available is broad, encompassing inhalers (Metered-Dose, Dry Powder and Aqueous Droplet) and nebulisers.
Due to the complex of the respiratory tree, the aerosolization performance of the inhalation drug product should be consistent, uniform and capable to deliver to the lung fine particles, less than 5 µm.
In-vitro experimental methods provide means for the quality control of existing Dry Powder Inhalers (DPIs), Metered-Dose Inhalers (MDIs, pressurized or non-pressurized), Aqueous Droplet Inhalers (or Soft Mist Inhalers) and nebulizers and for exploring the influence of formulation and device parameters. All products as described in the EU and US pharmacopoeia should be characterized for delivered dose uniformity and droplet/particle size distribution  .
 European Pharmacopoeia:
0671 Preparations for inhalation
2.9.44 Preparations for nebulisation: characterisation
2.9.18 Preparations for inhalation: aerodynamic assessment of fine particles
2.9.31 Particle size analysis by laser light diffraction
General Chapter <5> Inhalation and Nasal Drug Products - General Information and Product Quality Tests
Chapter <1151> Pharmaceutical Dosage Forms
Chapter <601> Product Performance Tests - Nasal, Inhalation, Aerosols, Sprays and Powders
Chapter <1601> Products For Nebulisation - Characterization Tests
N-nitrosoamines, refer to any molecule containing the nitroso functional group. These molecules are of concern because nitrosamine impurities are probable human carcinogens. The unexpected finding of nitrosamine impurities in drugs such as angiotensin II receptor blockers (ARBs), ranitidine, nizatidine, and metformin, has made clear the need for a risk assessment strategy for potential nitrosamines in any pharmaceutical product at risk for their presence. Stringent regulatory guidance´s are not only helping to make drugs safer, but through the method requirements are helping laboratories to identify the appropriate tools needed for accurate and confident analysis.
Since 2018 FDA  and EMA  have established fist risk assessment and provide guidance for analytical method to be use for the analysis of nitrosamines and on a second stage regulation and limit to be determine in certain kind of drugs.
Various analytical technique can be use for the scope. As several drugs have been recalled due to contamination with N-nitrosodimethylamine (NDMA), a probable human carcinogen. Dimethylamine (DMA) and nitrite are precursors in the formation of NDMA. Ion chromatography (IC) methods were developed for the determination of these two precursors in drug substances and drug products. Dimethylamine was determined by a cation exchange separation with suppressed conductivity detection using one of two methods.
The US FDA and EMA regulatory guidance on controlling the level of nitrosamine impurities in human drugs, have established then the needs to develop a highly sensitive and robust LCMS and GCMS solution for routine screening and batch testing of drug substances and products.
Analytical method for a reliable and sensitive target determination for nitrosamine need high performance in term of chromatography and mass spectrometry instruments. In this lecture we will describe which are the more appropriate technique to achieve those results and which are the advantage of each of those comparing chromatography approaches  and describing differences between triple quadrupole technologies  and Orbitrap technologies  for the MS point of view.
 https://www.ema.europa.eu/en/human-regulatory/post-authorisation/referral-procedures/nitrosamine-impurities Nitrosamine EMEA-H-A5(3)-1490
 D. Lu, D. Hower, A. Lamb, A. Romano , D. Roberts, C. Cojocariu Application Note 10753 Thermofisher Scientific
 H. Yang, E. Moy, M. Volny, C. Martins ,M. Du, W. Elmasri, T. Tadey ; Application note 74059 Thermifsher Scientific
 H. Yang, J. Bardsley, M. Du; Application Note 000362 Thermofisher Scientific.
The quality of every drug product is verified by strict laboratories´ analysis. The microbiological test on drug products are performed to verified the compliance to limits stated by Pharmacopeia, related to Total Aerobic Microbial Count, Total Yeast Mold Count and specific microorganism and to research of Endotoxin (LAL test method D- Chromogenic kinetic method). Every method used to verify the quality of drug products has to be validated: the suitability requires to inoculate the product with specific microorganism or endotoxin in order to challenge the analysis method.
The aim is to obtain a recovery of microorganism or endotoxin that guarantee the robustness of the test. With the purpose to guarantee the safety and compliance of the analysis an environmental monitoring according to Annex 1 is performed by analyzing the microbiological quality of lab surface and air. Microbiological suitability requires use of pathogen strains that has to be revivification and prepared according to specific procedure.
 EP currently official ed: par 2.6.12. Microbiological examination of non-sterile products: microbial enumeration tests and par 2.6.13. Microbiological examination of non-sterile products- test for specified micro-organisms
 USP currently official ed: par 61 Microbiological examination of non-sterile products: microbial enumeration tests and par 62 Microbiological examination of non-sterile products: tests for specified microorganisms.
 JP current edition: par 4.05 Microbial Limit Test  EP currently official ed: par. 2.6.14 Bacterial Endotoxin Test - method D (Chromogenic kinetic method)
 EP currently official ed: par. 5.1.10. GuideLines for using the test for bacterial endotoxins
 USP currently official ed: <85> Bacterial Endotoxins Test
 USP currently official ed: <1085> Guidelines on the endotoxin test
 EudraLex - Volume 4 - Good Manufacturing Practice (GMP) guidelines
In the manufacture of medicinal products and APIs, the cleaning of facilities and equipment is an important process to avoid contamination and cross contamination. In compliance with the GMP regulations, cleaning is performed and documented according to the described procedures. In the past, cleaning effectiveness was often monitored only visually. However, residuals of APIs and synthesis intermediates as well as of detergents are increasingly an issue in inspections and audits. The success of cleaning procedures has to be validated. In addition to the FDA and EU GMP, the PIC/S document PI 006, ANNEX 15, ICH Q7 and APIC address cleaning validation.
We will focus on: cleaning validation vs cleaning verification, risk index calculation and worst case determination, PDE determination and analytical method validation for residuals determination.
 EU GMP guideline chapter 3.3.6 - EMA/CHMP/CVMP/SWP169430/2012 - Guideline on setting health based exposure limits for the use in risk identification in the manufacture of different medicinal products in shared facilities
 Q&A EMA/288493/2018
 Q&A EMA/CHMP/CVMP/SWP/463311/2016
 EU GMP guideline chapter 3.1.5 and Annex 15
 FDA CFR 211.167 Equipment Cleaning and Maintenance
 FDA Guide to Inspection Validation of Cleaning Processes (7/93)
 EU GMP guideline Part II Basic requirements for Active Substances used as starting materials
 APIC: Guidance on Aspects of Cleaning Validation in Active Pharmaceutical Ingredients Plants, September 2016
 PIC/S - PI 006-3: Recommendations on cleaning validation (09/2007)
 ICH Q7 - GMP Guide for active pharmaceutical ingredients (11/2000)
During pharmaceutical development, safety, efficacy and performance of the pharmaceutical drug product need to be ensured. This requires a good understanding of the relevant Key Quality Attributes, which are then integrated into a control and specification strategy. Routine testing is further executed during release and stability studies.
In context of New Chemical Entities, general approaches are well established. Nevertheless, the chemical structures of the new Active Pharmaceutical Ingredients are getting more and more complex. This is translating typically into more challenging physico-chemical properties. This needs to be addressed by enabling formulation technologies, which are also getting more and more engineered.
Consequently, those formulations are often more sensitive to scalability during manufacturing, moisture ingress and storage time. In addition, to understand the impact of these relevant contributing factors, more and more analytical tools might be required to understand the underlying mechanisms of component interplay.
In this talk, a series of case studies will be presented, aiming to demonstrate the complementarity of analytical techniques in elaborating hypothesis that may highlight critical attributes or process parameters.
Those iterative learnings are required steps to have a better control and confidence on the robustness of these more complex engineered formulations.
Stability is a critical parameter for the pharmaceutical industries that affects the quality of drug products. Failure of stability leads to product recalls and may lead to loss of revenues for the sponsor. The stability profile of a New Chemical Entity  must be established during the entire product´ life-cycle. Stability studies must be conducted during drug discovery and product development, to support a marketing application and post-approval changes.
Stability data presented at the time of the regulatory submission should be combined with the knowledge gained during the product development phases to establish a product´ Shelf-Life.
The product quality and preservation of the therapeutic benefit should be demonstrated and monitored over the product´ shelf-life, under the influence of various environmental conditions, such as temperature, humidity and light either during manufacturing and storage. In these terms, also the interaction with the excipients within the formulation and the interactions with the packaging material gain an important role to maintain product stability.
ICH Stability Guidelines  provide the pharmaceutical companies with the storage conditions and the testing frequency to apply during the stability studies, however these studies are to be designed based on the intrinsic stability of the active molecule and its tendency to degrade. Additional stability studies may be useful during development to predict and evaluate the stability of the final pharmaceutical product to select the most appropriate container closure system, give the label recommendations for storage and set end-of-life specification.
 New Drugs at FDA: CDER´s New Molecular Entities and New Therapeutic Biological Products (FDA, 2020)
 ICH Q1A(R2): Stability Testing of new Drug Substances and Products, CPMP/ICH/2736/99, August 2003; and related ICH Q1B, Q1C, Q1D and Q1E
Development of an analytical method is crucial to test a defined characteristic of the drug substance or drug product against established acceptance criteria for that characteristic . The purpose of the method should reflect the phase of drug development i.e. desirable standards of performance to support preclinical studies, GMP stability studies and GMP batch release testing for early phase I/II and late phase III clinical trials, all the way to commercial. In other words, method development is a continuous process that progresses in parallel with the evolution of the drug product, and may be refined/expanded, based on increased API and drug product knowledges. ICH Q14  recommends two approaches to development (1) Traditional approach One-factor-at time experiments (OFAT) and (2) Enhanced approach Analytical Quality by Design (AQbD) while ICH Q2  provides a detailed validation strategy to demonstrate that the Analytical procedure is acceptable for the intended use and provide accurate and reliable results at every stage of development from pre-clinical trials through marketed products. Validation also provides evidence that systems and methods are suitable. This presentation describes the general approach towards the development of an analytical method (RP-HPLC with a UV detector) with a focus on Analytical Quality by Design (AQbD) from Workflow, Knowledge, Risk Assessment, Robustness to Control Strategy, the latter being defined prior to Validation. Typical validation parameters to be considered during validation of analytical method (accuracy, precision, specificity, limit of detection, limit of quantification, linearity range and robustness) as well as requirements from early to late phase validation will be outlined.
 FDA: Analytical Procedures and Methods Validation for Drugs and Biologics Guidance for Industry
 ICH Q14 describes the scientific principles for development, change management and submission requirements of analytical procedures for the minimal and enhanced approach.
 ICH Q2(R2) provides guidance for establishing, submitting, and maintaining evidence that an analytical procedure is fit for purpose (assuring drug quality).
The quality of drug substance and drug product is determined by their design, development, process controls, good manufacturing practice (GMP) controls, process validation, and by specifications applied to them throughout development and manufacturing. The specifications of critical quality attributes (CQAs) are thus one part of a total control strategy designed to ensure product quality and consistency. Typically, CQAs for a given product are defined early in development prospectively based on the quality target product profile of the biotherapeutic, and progressively refined as additional product knowledge is gained over time with extensive analytical characterization, animal studies, clinical experience, data from lots used for demonstration of manufacturing consistency and data from stability studies. ICH Q6B guideline defines specifications as the set of criteria to which a drug substance or drug product should conform to be considered acceptable for its intended use. The specifications are proposed and justified by the manufacturer and approved by regulatory authorities as conditions of approval.
 ICH Topic Q 6 B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.
 ICH Q8 (R2) Pharmaceutical development.
The quality of medicines is obtained by an holistic approach starting from product characterization, process development, quality of raw and starting materials, process validation and controls, product specifications and application of good manufacturing practices (GMP). Many tools are available to support manufacturing and regulatory activities aimed to deliver quality products to patients.
Guidelines [1,2] have been developed as harmonized and state-of-the-art indications, developed by the scientific community, to support product development and regulatory applications, while Pharmacopoeia monographs  represent mandatory standards to be applied to raw materials/starting materials/active substances/finished products. GMP requirements  have to be mandatorily applied by manufacturers of both investigational and approved medicinal products. Depending on the development stage, the regulatory requirements should be applied in an increasingly stringent way, taking into account the progressive gain of knowledge and the final indication and administration to patients. To this respect, above general GMP and scientific guidelines from EMA and ICH, specific guidelines have been developed to address the specific requirements of investigational medicinal products [5,6].
An example on requirements at different development stages on biotherapeutic products will be presented.
 ICH quality guidelines https://www.ich.org/page/quality-guidelines
 EMA scientific guidelines for Biologicals https://www.ema.europa.eu/en/human-regulatory/research-development/scientific-guidelines/biological-guidelines
 EudraLex - Volume 4 - Good Manufacturing Practice (GMP) guidelines https://health.ec.europa.eu/medicinal-products/eudralex/eudralex-volume-4_en
 European Pharmacopoeia 11th Edition
 EMA/CHMP/BWP/534898/2008 rev. 2: Guideline on the requirements for quality documentation concerning biological investigational medicinal products in clinical trials https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-requirements-quality-documentation-concerning-biological-investigational-medicinal_en-2.pdf
 Volume 4 EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use. Annex 13: Investigational Medicinal Products https://health.ec.europa.eu/system/files/2016-11/2009_06_annex13_0.pdf
Nuclear Magnetic Resonance (NMR) has been used, for a long time, for pure compounds analysis: typical uses are, for example, structural determination, regio-isomers identification, analysis of residual solvents and contaminants in reaction products, adulterants identification.
In recent years, thanks to the improvement of the technology, and the development of dedicated platforms, it has been recognized the extraordinary potential of NMR in the analysis of complex mixtures like biological fluids, foods, beverages, natural products, pharmaceutical formulations.
The study of these complex mixtures involves two different levels of analysis: the identification of the individual components (targeted) and the global measurements of the multiparametric response to external factors (untargeted).
The first level requires a selective approach, potentially enabling the identification and quantification of most metabolites in a complex matrix. The second level is rather based on statistical methods and databases of spectra.
Nuclear Magnetic Resonance is a powerful technique for complex samples profiling by means of both targeted and untargeted approach, which competes with traditional methods that usually require reference standards.
Moreover, the NMR technique is inherently quantitative, thus allowing a highly accurate determination of the concentration of single components in a mixture. Due to minimal sample preparation and the high-throughput automation, proton-NMR offers, in addition, a low-cost-per-sample screening.
In recent years, fragment-based screening (FBS) by NMR became also very popular. NMR is ideally suited for detecting low affinity ligands in primary screens. In addition, screening by NMR allows quality control of the screening library which makes NMR superior to other methods.
Some examples of applications will be shown.
Among the Gonadotropin Releasing Hormone (GnRH) modulators , elagolix represents a breakthrough being the first non-peptide orally active GnRH-antagonist approved for the treatment of sex-hormone dependent diseases such as endometriosis  and uterine fibroids . Chemically, it is an uracil-based derivative having a stereocenter with (R)-configuration and an additional source of chirality, called atropisomerism, arising from a restricted rotation around a C-C bond due to steric hindrance involving the o-fluorine of the 5-aryl group with the methyl and the carbonyl oxygen at 6- and 4-position of the uracil moiety, respectively (Figure 1).
Figure 1: Chemical structure of elagolix: the hindrance to rotation about the single bond is indicated by the black arrow.
Since atropisomerism occurs via a time/temperature-dependent bond rotation causing a conformational instability, it is very important to be considered in drug discovery and development processes.
Herein, we will focus on the investigation of the elagolix conformational stability, through spectroscopic, analytical, and theoretical techniques, and of few new analogues differently substituted at the 6- or 4-position of the uracil moiety. These derivatives showed atropisomeric interconversion rates lower than elagolix, allowing their separation and the analyses as single atropisomers.
Overall, these outcomes contributed to clarify the structural determinants involved in the control of the spatial arrangement of the substituents within this molecular framework, useful for future development of single atropisomers with higher selectivity.
 Casati L., Ciceri S., Maggi R., Bottai D. Biochem. Pharmacol. 2023, 212, 115553.
 Lamb Y. N. Drugs 2018, 78 (14), 1501-1508.
 Muhammad J., Yusof Y., Ahmad I., Norhayati M.N. BMC Womens Health 2022, 22(1), 14.
 Ciceri, S.; Colombo, D.; Fassi, E.M.A.; Ferraboschi, P.; Grazioso, G.; Grisenti, P.; Iannone, M.; Castellano, C.; Meneghetti, F. Molecules 2023, 28, 3861.
 Toenjes, S.T.; Gustafson, J.L. Atropisomerism in medicinal chemistry: challenges and opportunities. Future Med. Chem. 2018, 10, 409-422
Introduction and aim
Glycosylation is one of the most common protein post-translational modifications, regulating many biological and physiological processes, including intracellular trafficking, cell adhesion and the immune response . Alterations in the glycosylation pattern are often associated with malignant transformation, as they are involved in many aspects of tumor development and progression, including proliferation, invasion, angiogenesis and metastasis . The most widely occurring cancer-associated changes in glycosylation include an overall increase in sialylation, especially in a2,3'- and a2,6'-linked sialylation. Moreover, malignant cells are characterized by abnormal expression of truncated O-glycans, such as Thomsen-Friedenreich related antigens and their sialylated forms (T, Tn and sT, sTn, respectively). In particular, sTn has attracted widespread attention in the recent years, as it is expressed in 80% of human carcinomas, and absent or only weakly expressed in healthy tissues . For this reason, it can be considered a valuable cancer biomarker. However, sTn has historically been difficult to target with specificity and selectivity. As a matter of fact, glycan recognition represents a very tough challenge: the main issues are related to the fact that the binding probes have to be able to distinguish between a wide family of structures, including closely related isomers. Furthermore, the recognition process is hampered by the poor immunogenicity of glycans, due to their transient nature . To date, glycan analysis is mostly based on the use of antibodies and lectins. However, high costs, poor availability, low affinity and issues in storage and application conditions make such receptors limited . Molecular Imprinting technique can offer a promising alternative: Molecularly Imprinted Polymers (MIPs) are specific capture tools with tailor-made binding sites showing recognition abilities comparable to those of antibodies. MIPs are obtained through polymerization of functional monomers and of a crosslinker in presence of a template molecule which is eventually removed, leaving behind cavities exhibiting chemical and steric complementarity [6, 7]. MIPs can be produced in a variety of formats, but in the recent years biocompatible water-based nanogels (NGs) are gaining growing interest over the classical bulk-produced materials . Therefore, the aim of this PhD project was to develop a MIP-NG targeting the sTn antigen with specificity and selectivity.
The first part of the project aimed at optimizing the MIP-NG synthesis protocol and at developing reliable methods to quantify affinity, using sialylated disaccharades as model templates.
Reaction conditions were tuned to maximize the yield and a novel, elegant and accurate Affinity Capillary Electrophoresis (ACE) method was set up to monitor NGs-template interactions in free solution and to retrieve precise affinity parameters. A small library of MIPs was synthesized to evaluate applicability of three different functional monomers and to find the optimum composition. Finally, imprinting was carried out by using in-house synthesized glycopeptides as templates, to achieve a higher degree of resemblance with the natural occurring biomarker.
Sialyllactose-imprinted NGs: synthesis, characterization, stability testing and development of ACE method
NGs were first synthesized by adapting a well-established protocol where mixtures of polar and non-polar monomers were successfully used for imprinting via precipitation polymerization in water . Commercially available 3-acrylamidophenylboronic acid (3-AAPBA), along with an in-house synthesized amidinium derivative, were used as functional monomers [10, 11]. Model sugars (i.e., a2,3' and a2,6'-sialyllactose, 3SL and 6SL respectively) were used as templates to mimic the glycan moiety of the sTn antigen. After synthesis, NGs were characterized by Dynamic Light Scattering (DLS) in terms of size, polydispersity index (PDI) and Z-potential. Atomic Force Microscopy images of the NGs were also produced, in collaboration with Prof. Maddalena Patrini from the Department of Physics at the University of Pavia. NGs were found to be spherical and homogeneous, with a diameter ranging from 70 to 100 nm, and positively charged (average Z-potential +30 mV). Moreover, a stability study was performed in order to find the best storage protocol to make MIP-NGs suitable for long term applications : to this end, size, PDI and charge were monitored by DLS over time for materials stored at controlled room temperature (25 ºC) and in cold environment (4 ºC), both in absence and in presence of NaN3. Polymers were found to be stable at room temperature for at least 30 days. Further, storage in the fridge does not seem to affect gel properties, while size and PDI seem to increase upon addition of preservative, indicating a poor stability. In conclusion, to both prevent bacterial growth and particle aggregation, refrigeration was identified as the correct way to store the materials. Affinity towards template was preliminarily evaluated by classical batch rebinding studies by linking templates to magnetic beads to easily separate bound from free fraction. These experiments confirmed specificity and selectivity of MIPs. However, there are more accurate methods to quantify complexation: Affinity Capillary Electrophoresis (ACE) has gained increasing interest, as it can provide an extensive understanding of chemical interactions without the need of immobilization of the involved species . Therefore, Dynamic Complexation CE (DCCE) was selected to assess NGs recognition properties. After a proper optimization of CE conditions, including type, concentration and pH of running buffer, capillary length and sample preparation protocol, a novel method was set up: NGs migrated with a very sharp, reproducible and efficient peak. Polymers were then injected, first in a capillary filled with plain buffer and then in capillary filled with buffer added with increasing concentrations of template or analogue molecules. Peak mobility shift was recorded and plotted versus template concentration to obtain binding isotherms: Kd values for MIPs were found to be in the μM range, indicating a good affinity. Notably, selectivity was confirmed, indicating a successful imprinting.
Sialyllactose-imprinted NGs: polymer composition optimization
It was then decided to investigate more deeply the boronate chemistry, so to evaluate the best functional monomer in terms of affinity. In addition to 3-AAPBA, two other boronic acid derivatives were selected for this study: i) 2-acrylamidophenylboronic acid (2-AAPBA), which is supposed to provide better performances due to intramolecular coordination between boron and oxygen atoms , ii) (acrylamidophenyl)benzoboroxole (AAmBOB), belonging to the benzoboroxole receptors showing improved affinity for glucuronates . NGs were synthesized by using the protocol initially established, and 3SL and 6SL were used as templates. Non-imprinted polymers were also synthesized as negative controls. After a full DLS characterization, recognition abilities of the different NGs series were assessed by ACE using the optimized method: first, the three different 6SL-MIPs were injected in buffer added with increasing concentrations of their own template (6SL), to evaluate the ability of boronate functional monomers to complex the sialyllactose, then affinity of all NGs towards both templates (6SL and 3SL) was investigated. A very large shift, even at very low concentrations, was observed for 2-AAPBA NGs, while AAmBOB NGs exhibit poor recognition for their own template; 3-AAPBA NGs showed intermediate shift values. Therefore, results confirmed what expected and 2-AAPBA was selected for subsequent studies.
Glycopeptide-imprinted NGs: synthesis, characterization and affinity testing
Although sialyllactose represents a good model for sialylation in cancer, it does not reflect the complexity of the naturally occurring biomarker. For this reason, SLs were attached to a customized pentapeptide sequence from MUC1, whose abnormal glycosylation has extensively been correlated with cancer transformation . Glycosylation of the peptide was performed via reductive amination and the product was purified by an optimized HPLC method, then protective groups were properly removed. NGs were synthesized by applying the optimized protocol and were characterized by DLS in terms of size, PDI and charge. Compared to SLs-gels, dimensions were found to be slightly higher, but still lower than 150 nm. PDI was in the acceptable range and Z-potential was found to be strongly positive (+45 mV), indicating a stable particle suspension. Affinity of these new NGs was first evaluated by batch rebinding: protocol was based on immobilization of polymers on a polystyrene 96-well microplate, followed by incubation with templates and quantification of the unbound fraction by spectroscopic measurements. Results confirmed a successful imprinting, as the control gels showed the weakest binding, while MIPs exhibited higher affinity for their own templates. To further validate specificity of polymers, recognition of the non-glycosylated peptide was tested as well: Kd values still indicate some binding, but the extent is lower. Moreover, a preliminary Surface Plasmon Resonance (SPR) method was set up: glycopeptide templates were immobilized on a gold chip and increasing concentrations of NGs were injected. Again, specificity was confirmed, and binding isotherms showed a Langmuir-like trend. Finally, ACE was used to further verify NGs performances in terms of capture abilities.
As an overall conclusion, during this PhD project, novel MIPs in NG format were designed to target simplified versions of tumor-specific glycoprotein biomarkers.
First, NGs were synthesized using commercially available monomers and model sugars as templates, and a full characterization, including a stability test, was carried out. These polymers were used to set up for the first time an ACE method to accurately quantify the recognition abilities. This technique proved to be a very powerful analytical platform to monitor dynamic polymer-template complexation and was also very useful to tune the composition of the MIPs in terms of functional monomers to choose the best binder for the selected templates. Then, the tested sugars were linked to a pentapeptide sequence to be used as new template to better mimic the natural occurring cancer-related antigen. The newly synthesized NGs were tested for affinity with multiple analytical approaches, including SPR, which confirmed their specificity and selectivity.
Even if further optimization of the synthesis protocol is necessary to achieve the optimum conditions and to accomplish the targeting of the actual biomarker, this work represents an important step forward in glycan recognition and a valuable proof of concept of this kind of diagnostic tools. Moreover, it strongly underlines the importance of developing analytical methods to get reliable and robust affinity data, in a future perspective of cell-based test applications and ultimately of clinical applications.
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Oxidative stress and chronic inflammation are two phenomena that are directly involved in various diseases, from metabolic to neurological disorders and even in the formation of tumor . In this context, a wide source of bioactive molecules exhibiting anti-inflammatory effects, combined with antioxidant potential, can be found in natural extracts . Whereas previously, as a source of these phytochemicals, the focus was on the 'nobler' parts of the plant, i.e. the fruits, leaves and roots, today much emphasis is placed on investigating by-products from the agribusiness production chain. Specifically, this strategy aimed at the reuse of industrial by-products could reduce the impact of cultivation on waste production, minimizing disposal problems, while obtaining a potential source of bioactive compounds.
In particular, it has recently been reported that certain phytochemicals are able to target two closely related systems within the cell, namely the NRF2 and NF-kb pathways , . While the NRF2 pathway is responsible for regulating an extensive panel of antioxidant enzymes, NF-kb is the main effector pathway involved in inflammation. Phytochemicals in general mediate their effects by modulating the DNA-binding capacity of both these transcriptional factors and, in turn, correcting the state of cellular imbalance related to an uncontrolled inflammatory condition. In particular, apple-specific polyphenols exert antioxidant, anti-inflammatory and lipid-lowering effects due to the components contained in every part of the fruit (peel, stark, seeds and pulp). This biological activity was also confirmed in preclinical studies in which isolated apple polyphenols (AP) were shown to be effective in the prevention/treatment of various metabolic and inflammatory diseases, from to hypercholesterolemia  to ulcerative colitis .
Since apples are one of the most consumed fruits in the world, the related waste generated at all stages of the agricultural process has a great impact on the environment. The thinning process in particular, i.e. the removal and dumping of young apples one month after blossom to guarantee the quality of the harvested ripe apples, heavily contributes on the generation of waste of potentially bioactive material. Young apples, in fact, are particularly rich in polyphenols (almost 10-fold compared to harvested ripe apples), and could be exploited as such .
During the first two years of my PhD, I mainly focused on the qualitative profiling of a thinned apple polyphenol extract (TAP) using LC-HRMS (positive and negative ionic mode) approaches, followed by the exploration of its potential anti-inflammatory and antioxidant activity using multiple cell-based assays and the latest proteomics strategies; main results were published in a peer-reviewed article in 2022 . Figure 1 illustrates and resumes the preject design.
Figure 1: Graphical representation of the different approaches used to define the qualitative profile and to evaluate the biological activity of thinned apple polyphenols (TAP) extract.
A targeted approach was used to identify 52 compounds using an in-house library of potential apple metabolites, while 16 were identified by an untargeted approach based on computing the elemental composition of each newfound ion, used to search an online database that generates probable candidates from the chemical formula; in turn, each candidate has to be validated and, only if certain, identified as being present in the extract. Of a total of 68 compounds, 65 were polyphenols; 39 compounds commonly detected in both polarities, while 7 were unique in negative ion mode, and 6 in positive. In detail, among the 52 compounds identified by the targeted approach, 20 were phenolic and organic acids, 11 flavanols, 10 flavonols, 5 flavanones, 4 dihydrochalchones, 1 flavone (luteolin) and 1 triterpenoid (euscaphic acid). The 16 compounds identified using the untargeted approach are instead classified as follows: 9 flavonols, 3 phenolic acids, 1 flavanone, 1 dihydrochalchone and 2 lipids.
Once the concentration of total polyphenols in the TAP extract had been determined, the radical scavenging activity of the extract was assessed using the DPPH assay, revealing an activity significantly higher than the reference compounds when expressed on the basis of polyphenol content. The radical scavenging activity determined suggests a potential direct antioxidant activity of the extract, although the most accepted hypothesis to date refers to an indirect antioxidant defense mechanism involving the activation of the NRF2 pathway; moieties such as ortho-diphenols, which characterize some polyphenols, once oxidized to the corresponding quinones in an environment characterized by a marked oxidative stress, will acquire a more electrophilic character that will allow these molecules to interact with the highly reactive cysteines of the Kelch-like ECH-associated protein 1 (KEAP1); the latter interacts with NRF2 in a redox-sensitive manner and the dissociation of the proteins in the cytoplasm is followed by transportation of NRF2 to the nucleus.
This hypothesis was confirmed by treating a NRF2/ARE Responsive Luciferase Reporter HEK293 stable cell line treated with increasing concentrations of TAP extract (1 - 250 μg/mL) for 6 or 18 hours. After 6 hours, the effect started to be significant at a concentration of 50 μg/mL to induce a 2.3-fold increase at a 250 μg/mL concentration. The fold increase was higher after an incubation time of 18 hours to reach more than a 5-fold increase at the highest tested dose. In parallel, the extract (TAP) was also tested on a NF-kb Responsive Luciferase Reporter R3/1 stable cell line challenged with two different pro-inflammatory agents: TNF- α and Il-1 α for 6 hours or 24 hours, respectively. NF-kB induced luciferase was dose-dependently reduced by TAP pre-treatment (16 hours).
In order to acquire information concerning the impact of the phytocomplex on the proteome of a cellular model of inflammation, a quantitative proteomic study was conducted using both a SILAC (Stable Isotope Labelling by Amino acids in Cell culture) and a LFQ (label-free quantitative) approaches. The experiment was designed to evaluate the effect of TAP on the cellular proteome under homeostatic conditions and thus to exclude any toxicity effect, and to test its efficacy under a pro-inflammatory stimulus-induced oxidative stress condition. Although the two approaches are characterized by different levels of performance (e.g., different number of proteins identified and significantly quantified), the same modulated cellular pathways were identified, making the results more robust.
As expected, many up-regulated genes involved in the NRF2 pathway activation were detected. Besides, the crucial finding is that TAP treatment over-expresses the inducible isoform of heme oxygenase (HMOX1), a well-established immunomodulator ; the induction of HMOX1 protects against the cytotoxicity caused by oxidative stress and apoptotic cell death, making HMOX1 an appealing target for the treatment of several chronic inflammatory diseases , .
In conclusion, these first results, suggested that thinned apples can be effectively considered a valuable source of apple polyphenols to be used in health care products to prevent/treat oxidative and inflammatory chronic conditions.
To critically evaluate if the extract could efficiently dampen an inflammatory condition in vivo, the extract was orally administered (10 mg/kg) in a murine DNBS model of ulcerative colitis (UC). The incidence rate of UC, one of the Inflammatory Bowel Diseases (IBDs), has been increased in the last decades . Although the etiology is still unclear, the most accepted hypothesis centers on defects in tight junctions that result in increased intestinal permeability, leading to increased entry of luminal antigens that contribute to intestinal inflammation. Currently available drug therapies represent symptomatic treatment, with the goal of keeping patients in a state of remission to avoid flare-ups, which greatly affect his or her quality of life. Nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids succeed in partially relieving the symptoms of the disease and, to date, are considered the drugs of choice for the treatment of ulcerative colitis, although they are associated with non-negligible side effects. Hence the interest in finding new therapeutic strategies that counteract inflammation as effectively without inducing damage in other body districts. Since oxidative stress and inflammation contribute to tissue damage during colitis, the administration of natural compounds with antioxidant and anti-inflammatory activity has recently been proposed as a treatment for IBD. Apple polyphenols for instance are effective compounds in inhibiting microbial dysbiosis, chronic inflammation, and modulating intestinal permeability, and could therefore be used as dietary supplements to improve gut health. Given the characteristics of thinned apple by-product and with a view to sustainability, we moved to evaluate the therapeutic potential of TAP extract.
This project was carried out during my third year of PhD in collaboration with the University of Messina which handled the treatment of the animals (mice) and collected the colon tissue after sacrifice.
Using state of the art techniques in the field of proteomics we performed ex vivo functional studies to describe, at a molecular level, the biological processes distinctive for the pathological phenotype, and to understand the molecular pathways evoked by TAP extract to effectively limit the inflammatory state. In detail, more than 5400 proteins were identified and quantified in colon tissues from to different experimental groups, according to the canonical label-free quantitative (LFQ) proteomic approach as previously described . These data were then interpreted and rationalized using gene ontology annotation-based software, such as STRING and Ingenuity Pathway Analysis.
Overall, proteomics studies confirmed the usefulness of the chosen animal model characterized by immune cells infiltration and marked oxidative stress, and outlined the following molecular pathways evoked by TAP treatment: (i) activation of antioxidant-acting mechanisms; (ii) reversal of mechanisms overexpressed/activated in the presence of DNBS, with particular reference to mechanisms of ferroptosis and heme-toxicity; (iii) inhibition of the immune response; (iv) down-regulation of agents implicated in wound healing, as a reduced ulcerative condition may have generated less tissue damage and, consequently, less need for activation of coagulation, inflammation and angiogenesis processes. A summary of these results is reported in Figure 2 below.
Figure 2: Graphical representation of the main results from the quantitative proteomic study, from top to bottom the hallmarks of the DNBS-induced disease state and the effect of TAP extract reverting this condition.
Taken together, these results suggest that thinned apples may be considered a valuable source of polyphenols for use in health products to prevent/treat chronic inflammatory conditions; nevertheless, thinned apples represent a novel and as yet unexplored waste product for industrial production of bioactive extracts with significant economic and environmental impacts.
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