Dimethyl carbonate as a green alternative to acetonitrile in reversed-phase liquid chromatography. Part II: Purification of a therapeutic peptide.

Preparative liquid chromatography in reversed phase conditions (RPLC) is the most common approach adopted in the downstream processing for the purification of therapeutic peptides at industrial level. Due to the strict requirements on the quality imposed by the Regulatory Agencies, routinary methods based on the use of aqueous buffers and acetonitrile (ACN) as organic modifier are commonly used, where ACN is practically the only available choice for the purification of peptide derivatives. However, ACN is known to suffers of many shortcomings, such as drastic shortage in the market, high costs and, most importantly, it shows unwanted toxicity for human health and environment, which led it among the less environmentally friendly ones. For this reason, the selection of a suitable alternative becomes crucial for the sustainable downstream processing of peptides and biopharmaceuticals in general. In this paper, a promising green solvent, namely dimethyl carbonate (DMC) has been used for the separation of a peptide not only in linear conditions but also for its purification through non-linear overloaded chromatography. The performance of the process has been compared to that achievable with the common method where ACN is used as organic modifier and to that obtained with two additional solvents (namely ethanol and isopropanol), already used as greener alternatives to ACN. This proof-of-concept study showed that, thanks to its higher elution strength, DMC can be considered a green alternative to ACN, since it allows to reduce method duration while reaching good purities and recoveries. Indeed, at a target purity fixed to 98.5 %, DMC led to the best productivity with respect to all the other solvents tested, confirming its suitability as a sustainable alternative to ACN for the purification of complex biopharmaceutical products.

Assessing the performance of new chromatographic technologies for the separation of peptide epimeric impurities: the case of Icatibant.

The biopharmaceutical industry faces the challenge of efficiently characterising impurity profiles of therapeutical peptides, also due to their complex polar and ionisable attributes. This research explores the potential of advanced chromatographic techniques to address this challenge. The study compares dynamic electrostatic repulsion reversed phase (d-ERRP) to its counterparts (static ERRP and ion pair reversed phase IP-RP) in analysing Icatibant and its elusive epimeric impurity, [L-Arg]1-Icatibant and highlights its exceptional capabilities in generating symmetric peaks, mitigating the common tailing phenomenon, and serving as a steadfast guardian of column longevity. The result highlights d-ERRP as a pioneering tool in the domain of liquid chromatography, fostering its role as a reference technique for the analysis of therapeutic peptides.

Investigation of the GnRH antagonist degarelix isomerization in biological matrices.

One of the main objectives of peptide drug design is the improvement of peptide pharmacokinetics with maintaining biological activity, which can be achieved by the complex modifications of the primary structure of the peptides. However, these changes often lead to the formation of peculiar impurities in the peptide drugs and their metabolites, which require the development of advanced analytical methods to properly assess their content. Here, we investigated the degradation of the potent long-acting GnRH antagonist degarelix in various biologic media by the tailor-made HPLC method, which allows precise determination of 5-Aph(Hyd)-degarelix isomer, an impurity found in the degarelix active pharmaceutical ingredient (API) during its manufacturing. Unexpectedly, we discovered a rapid and irreversible conversion of degarelix API into the corresponding hydantoin isomer in serum, suggesting that this impurity can be also a potential drug metabolite in vivo. This finding underlines the importance of the development of more accurate and performing analytical techniques to correctly characterize the chemical composition of the manufactured drugs and their behavior under physiological conditions.

Discovery of Novel Allosteric EGFR L858R Inhibitors for the Treatment of Non-Small-Cell Lung Cancer as a Single Agent or in Combination with Osimertinib.

Addressing resistance to third-generation EGFR TKIs such as osimertinib via the EGFRC797S mutation remains a highly unmet need in EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we present the discovery of the allosteric EGFR inhibitor 57, a novel fourth-generation inhibitor to overcome EGFRC797S-mediated resistance in patients harboring the activating EGFRL858R mutation. 57 exhibits an improved potency compared to previous allosteric EGFR inhibitors. To our knowledge, 57 is the first allosteric EGFR inhibitor that demonstrates robust tumor regression in a mutant EGFRL858R/C797S tumor model. Additionally, 57 is active in an H1975 EGFRL858R/T790M NSCLC xenograft model and shows superior efficacy in combination with osimertinib compared to the single agents. Our data highlight the potential of 57 as a single agent against EGFRL858R/C797S and EGFRL858R/T790M/C797S and as combination therapy for EGFRL858R- and EGFRL858R/T790M-driven NSCLC.

Carbodiimide-Mediated Beckmann Rearrangement of Oxyma-B as a Side Reaction in Peptide Synthesis.

The suppression of side reactions is one of the most important objectives in peptide synthesis, where highly reactive compounds are involved. Recently, the violuric acid derivative Oxyma-B was introduced into peptide synthesis protocols as a promising additive to efficiently control the optical purity of the amino acids prone to racemization. However, we discovered a side reaction involving the Beckmann rearrangement of Oxyma-B during the coupling reaction, which compromises the yield and purity of the target peptides. Here, we present the investigation of the mechanism of this rearrangement and the optimization of the coupling reaction conditions to control it. These results can be taken into account for the design of novel efficient oxime-based coupling reagents.

Downstream Processing of Therapeutic Peptides by Means of Preparative Liquid Chromatography.

The market of biomolecules with therapeutic scopes, including peptides, is continuously expanding. The interest towards this class of pharmaceuticals is stimulated by the broad range of bioactivities that peptides can trigger in the human body. The main production methods to obtain peptides are enzymatic hydrolysis, microbial fermentation, recombinant approach and, especially, chemical synthesis. None of these methods, however, produce exclusively the target product. Other species represent impurities that, for safety and pharmaceutical quality reasons, must be removed. The remarkable production volumes of peptide mixtures have generated a strong interest towards the purification procedures, particularly due to their relevant impact on the manufacturing costs. The purification method of choice is mainly preparative liquid chromatography, because of its flexibility, which allows one to choose case-by-case the experimental conditions that most suitably fit that particular purification problem. Different modes of chromatography that can cover almost every separation case are reviewed in this article. Additionally, an outlook to a very recent continuous chromatographic process (namely Multicolumn Countercurrent Solvent Gradient Purification, MCSGP) and future perspectives regarding purification strategies will be considered at the end of this review.

Expanding the Use of Dynamic Electrostatic Repulsion Reversed-Phase Chromatography: An Effective Elution Mode for Peptides Control and Analysis.

Bioactive peptides are increasingly used in clinical practice. Reversed-phase chromatography using formic or trifluoroacetic acid in the mobile phase is the most widely used technique for their analytical control. However, sometimes it does not prove sufficient to solve challenging chromatographic problems. In the search for alternative elution modes, the dynamic electrostatic repulsion reversed-phase was evaluated to separate eight probe peptides characterised by different molecular weights and isoelectric points. This technique, which involves TBAHSO4 in the mobile phase, provided the lowest asymmetry and peak width at half height values and the highest in peak capacity (about 200 for a gradient of 30 min) and resolution concerning the classic reversed-phase. All analyses were performed using cutting-edge columns developed for peptide separation, and the comparison of the chromatograms obtained shows how the dynamic electrostatic repulsion reversed-phase is an attractive alternative to the classic reversed-phase.

From batch to continuous chromatographic purification of a therapeutic peptide through multicolumn countercurrent solvent gradient purification.

A twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process has been developed for the purification of a therapeutic peptide, glucagon, from a crude synthetic mixture. This semi-continuous process uses two identical columns operating either in interconnected or in batch mode, thus enabling the internal recycle of the portions of the eluting stream which do not comply with purity specifications. Because of this feature, which actually results in the simulated countercurrent movement of the stationary phase with respect to the mobile one, the yield-purity trade-off typical of traditional batch preparative chromatography can be alleviated. Moreover, the purification process can be completely automatized. Aim of this work is to present a simple procedure for the development of the MCSGP process based on a single batch experiment, in the case of a therapeutic peptide of industrial relevance. This allowed to recover roughly 90% of the injected glucagon in a purified pool with a purity of about 90%. A comparison between the performance of the MCSGP process and the classical single column batch process indicates that percentage increase in the recovery of target product is +23% when transferring the method from batch conditions to MCSGP, with an unchanged purity of around 89%. This improvement comes at the expenses of a reduction of about 38% in productivity.

A new sensitive and subunit-selective molecular tool for investigating protein kinase A in the brain.

Despite cellular complexity, a limited number of small molecules act as intracellular second messengers. Protein kinase A (PKA) is the main transducer of the information carried by cyclic adenosine monophosphate (cAMP). Recently, cellular imaging has achieved major technical advancements, although the search for more specific and sensitive low-molecular-weight probes to explore subcellular events involving second messengers is still in progress. The convergent synthesis of a novel, fluorescent small molecule comprising the cAMP structure and a rhodamine-based fluorescent residue, connected through a flexible linker, is described here. The interaction motif of this compound with PKA was investigated in silico using a blind docking approach, comparing its theoretical binding energy with the one calculated for cAMP. Moreover, the predicted pharmacokinetic properties were also computed and discussed. The new probe was tested on three areas of the mouse central nervous system (parietal cerebral cortex, hippocampus, and cerebellar cortex) with different fixation methods demonstrating remarkable selectivity towards the PKA RIα subunit. The probe showed overall better performances when compared to other commercially available fluorescent cAMP analogues, acting at lower concentrations, and providing stable labeling.

Modeling the nonlinear behavior of a bioactive peptide in reversed-phase gradient elution chromatography.

The thermodynamic behavior of octreotide, a cyclic octapeptide with important pharmaceutical functions, has been simulated under reversed-phase gradient elution conditions. To this end, adsorption behavior was firstly investigated in isocratic conditions, under a variety of water/acetonitrile + 0.02% (v/v) trifluoroacetic acid (TFA) mixtures as mobile phase by using a Langmuir isotherm. Organic modifier was varied in the range between 23 and 28% (v/v). Adsorption isotherms were determined by means of the so-called Inverse Method (IM) with a minimum amount of peptide. The linear solvent strength (LSS) model was used to find the correlation between isotherm parameters and mobile phase composition. This study contributes to enlarge our knowledge on the chromatographic behavior under nonlinear gradient conditions of peptides. In particular, it focuses on a cyclic octapeptide.

Production of peptides as generic drugs: a patent landscape of octreotide.

INTRODUCTION: New low-cost strategies and enhancement of the already described methods to manufacture peptide molecules on an industrial scale are highly requested, particularly for peptides such as octreotide, which, along with goserelin and leuprolide, dominate the global peptide market. A number of patents related to the production of octreotide can be found, concerning both solution and solid-phase synthesis. Thus, there is a need to revise the existing synthetic approaches in order to organize them in a more comprehensible way. AREA COVERED: The octreotide patent landscape could help improvement of the methods for manufacturing of octreotide in industrial scale, leading to the appearance of innovative approaches. EXPERT OPINION: The pharmaceutical value of octreotide can be seen from its high market percentage among other peptide drugs. The complex chemical structure of octreotide represents the main challenge for its industrial production. Two synthetic steps are crucial in the preparation of octreotide: (i) threoninol attachment or on resin formation working in solid-phase and (ii) disulphide bond formation to achieve cyclic structure. Analysis of various patents filed to date allows us to see the trend in simplification of the synthetic approaches from the labor intensive syntheses in solution to the more versatile and rapid solid-phase methods.

Common immunogenetic profile in children with multiple autoimmune diseases: the signature of HLA-DQ pleiotropic genes.

Type 1 diabetes mellitus (T1DM), celiac disease (CD) and autoimmune thyroid disease (ATD) are autoimmune conditions relatively common in paediatric age and frequently occur in association in the same subject. This event is not by chance and requires an explanation. Here, we studied the distribution of HLA-DQ αß heterodimers in 334 Italian children with T1DM, ATD and CD alone or in association and in 224 Italian healthy controls. In particular, 164 patients had T1DM (133 alone, 20+ATD, 7+CD and 4+CD+ATD), 118 had ATD (110 alone, 8+CD) and 52 had CD (40 alone, 11+ATD and 1+T1DM). 51 patients suffered from multiple autoimmune diseases. The risk for multiple autoimmune diseases was significantly associated with the increased number of HLA-DQ markers of susceptibility for both T1DM (p = 0.003) and CD (p = 0.006). The presence of one or more diabetogenic DQ molecules significantly increased the probability of developing not only T1DM (p < 0.001) but also CD (p < 0.001) and ATD (p = 0.001). Similarly, the presence of one or more celiac HLA-DQ heterodimers significantly increased the likelihood of developing not only CD (p < 0.001), but also T1DM (p < 0.001) and ATD (p < 0.001). We confirm that the sharing of the immunogenetic background is responsible for the development of multiple autoimmune diseases although with a different risk according to the number and type of susceptible HLA-DQ heterodimers as reported in the algorithm proposed here. It is likely that combinations of DQA1 and DQB1 alleles are the real culprits of the progression towards multiple autoimmune diseases and HLA-DQ genomic typing will improve the capability to predict associated autoimmune diseases in infancy.

Design of photocontrolled RNA-binding peptidomimetics.

Positively constrained: the first examples of photocontrolled RNA binding peptides are described. The large number of positively charged sides chains in the Rev response element (RRE) of an HIV type I targeting α-helix imposes constraints on the choice of azobenzene-derived crosslinker.

Tuning G-quadruplex vs double-stranded DNA recognition in regioisomeric lysyl-peptidyl-anthraquinone conjugates.

Anthraquinone is a versatile scaffold to provide effective DNA binders. This planar system can be easily conjugated to protonable side chains: the nature of the lateral groups and their positions around the tricyclic moiety largely affect the DNA recognition process in terms of binding affinity and mode, as well as sequence and structure of the target nucleic acid. Starting from an anthracenedione system symmetrically functionalized with N-terminal lysyl residues, we incremented the length of side chains by introducing a Gly, Ala, or Phe spacer, characterized by different flexibility, lipophilicity, and bulkiness. Moreover, 2,6, 2,7, 1,8, and 1,5 regioisomers were examined to yield a small bis(lysyl-peptidyl) anthracenedione library. By merging spectroscopic, enzymatic, and cellular results, we showed that the proper combination of a basic aminoacid (Lys) with a more hydrophobic residue (Phe) can provide selective G-quadruplex recognition, in particular when side chains are located at positions 2,6 or 2,7. In fact, while these derivatives effectively bind G-quadruplex structures, they behave at the same time as rather poor double-stranded DNA intercalators. As a result, the Lys-Phe substituted anthraquinones are poorly cytotoxic but still able to promote a senescence mechanism in cancer cells. This combination of chemical and biological properties foresees potentially valuable applications in anticancer medicinal chemistry.

Electromotive instillation of mitomycin immediately before transurethral resection for patients with primary urothelial non-muscle invasive bladder cancer: a randomised controlled trial.

BACKGROUND: The clinical effect of intravesical instillation of chemotherapy immediately after transurethral resection of bladder tumours (TURBT) has recently been questioned, despite its recommendation in guidelines. Our aim was to compare TURBT alone with immediate post-TURBT intravesical passive diffusion (PD) of mitomycin and immediate pre-TURBT intravesical electromotive drug administration (EMDA) of mitomycin in non-muscle invasive bladder cancer. METHODS: We did a multicentre, randomised, parallel-group study in patients with primary non-muscle invasive bladder cancer in three centres in Italy between Jan 1, 1994, and Dec 31, 2003. Patients were randomly assigned to receive treatment by means of stratified blocked randomisation across six strata. Patients and physicians giving the interventions were aware of assignment, but it was masked from outcome assessors and data analysts. Patients were randomly assigned to receive TURBT alone, immediate post-TURBT instillation of 40 mg PD mitomycin dissolved in 50 mL sterile water infused over 60 min, or immediate pre-TURBT instillation of 40 mg EMDA mitomycin dissolved in 100 mL sterile water with intravesical 20 mA pulsed electric current for 30 min. Our primary endpoints were recurrence rate and disease-free interval. Analyses were done by intention to treat. Follow-up for our trial is complete. This study is registered with ClinicalTrials.gov, number NCT01149174. FINDINGS: 124 patients were randomly assigned to receive TURBT alone, 126 to receive immediate post-TURBT PD mitomycin, and 124 to receive immediate pre-TURBT EMDA mitomycin. 22 patients were excluded from our analyses because they did meet our eligibility criteria after TURBT: 11 had stage pT2 disease and 11 had carcinoma in situ. Median follow-up was 86 months (IQR 57-125). Patients assigned to receive EMDA mitomycin before TURBT had a lower rate of recurrence (44 [38%] of 117) than those assigned to receive PD mitomycin after TURBT (70 [59%] of 119) and TURBT alone (74 [64%] of 116; log-rank p<0·0001). Patients assigned to receive EMDA mitomycin before TURBT also had a higher disease-free interval (52 months, IQR 32-184) than those assigned to receive PD mitomycin after TURBT (16 months, 12-168) and TURBT alone (12 months, 12-37; log-rank p<0·0001). We recorded persistent bladder symptoms after TURBT in 18 (16%) of 116 patients in the TURBT-alone group (duration 3-7 days), 37 (31%) of 119 in the PD mitomycin post-TURBT group (duration 20-30 days), and 24 (21%) of 117 in the EMDA mitomycin pre-TURBT group (duration 7-12 days); haematuria after TURBT in eight (7%) of 116 patients in the TURBT-alone group, 16 (13%) of 119 in the PD mitomycin post-TURBT group, and 11 (9%) of 117 in the EMDA mitomycin pre-TURBT group; and bladder perforation after TURBT in five (4%) of 116 patients in the TURBT-alone group, nine (8%) of 119 in the PD mitomycin post-TURBT group, and seven (6%) of 117 in the EMDA mitomycin pre-TURBT group. INTERPRETATION: Intravesical EMDA mitomycin before TURBT is feasible and safe; moreover, it reduces recurrence rates and enhances the disease-free interval compared with intravesical PD mitomycin after TURBT and TURBT alone. FUNDING: None.

Synthesis and antitumor molecular mechanism of agents based on amino 2-(3',4',5'-trimethoxybenzoyl)benzo[b]furan: inhibition of tubulin and induction of apoptosis.

Induction of apoptosis is a promising strategy that could lead to the discovery of new molecules active in cancer chemotherapy. This property is generally observed when cells are treated with agents that target microtubules, dynamic structures that play a crucial role in cell division. Small molecules such as benzo[b]furans are attractive as inhibitors of tubulin polymerization. A new class of inhibitors of tubulin polymerization based on the 2-(3',4',5'-trimethoxybenzoyl)benzo[b]furan molecular skeleton, with the amino group placed at different positions on the benzene ring, were synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell-cycle effects. The methoxy substitution pattern on the benzene portion of the benzo[b]furan moiety played an important role in affecting antiproliferative activity. In the series of 5-amino derivatives, the greatest inhibition of cell growth occurred if the methoxy substituent is placed at the C6 position, whereas C7 substitution decreases potency. The most promising compound in this series is 2-(3',4',5'-trimethoxybenzoyl)-3-methyl-5-amino-6-methoxybenzo[b]furan (3 h), which inhibits cancer cell growth at nanomolar concentrations (IC(50) =16-24 nM), and interacts strongly with tubulin by binding to the colchicine site. Sub-G(1) apoptotic cells in cultures of HL-60 and U937 cells were observed by flow cytometric analysis after treatment with 3 h in a concentration-dependent manner. We also show that compound 3 h induces apoptosis by activation of caspase-3, -8, and -9, and this is associated with cytochrome c release from mitochondria. The introduction of an α-bromoacryloyl group increased antiproliferative activity with respect to the parent amino derivatives.

Convergent synthesis and biological evaluation of 2-amino-4-(3',4',5'-trimethoxyphenyl)-5-aryl thiazoles as microtubule targeting agents.

Combretastatin A-4, a potent tubulin polymerization inhibitor, caused us to synthesize a novel series of 2-amino-4-(3',4',5'-trimethoxyphenyl)-5-aryl thiazoles with the goal of evaluating the effects of substituents on the phenyl at the 5-position of the thiazole skeleton on biological activities. An ethoxy group at the para-position produced the most active compound in the series, with IC(50) values of 0.03-0.9 nM against five of seven cancer cell lines. The most active compounds retained full activity in multidrug resistant cancer cells and acted through the colchicine site of tubulin. Treated cells were arrested in the G2/M phase of the cell cycle, with cell death proceeding through an apoptotic pathway that was only partially caspase-dependent. Preliminary results suggest that, in addition to cell death by apoptosis, cells were also killed via mitotic catastrophe as an alternative cell death mechanism.

Electron paramagnetic resonance (EPR) study of spin-labeled camptothecin derivatives: a different look of the ternary complex.

Camptothecin (CPT) derivatives are clinically effective poisons of DNA topoisomerase I (Top1) able to form a ternary complex with the Top1-DNA complex. The aim of this investigation was to examine the dynamic aspects of the ternary complex formation by means of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR). Two semisynthetic CPT derivatives bearing the paramagnetic moiety were synthesized, and their biological activity was tested. A 22-mer DNA oligonucleotide sequence with high affinity cleavage site for Top1 was also synthesized. EPR experiments were carried out on modified CPT in the presence of DNA, of Top1, or of both. In the last case, a slow motion component in the EPR signal appeared, indicating the formation of the ternary complex. Deconvolution of the EPR spectrum allowed to obtain the relative drug amounts in the complex. It was also possible to demonstrate that the residence time of CPT "trapped" in the ternary complex is longer than hundreds of microseconds.

Epithelioid hemangioendothelioma of the facial bone.

The epithelioid hemangioendothelioma is an uncommon vascular neoplasm. Numerous sites of involvement are possible, but these tumors most commonly arise in the soft tissues, liver, and lung. We report a case of epithelioid hemangioendothelioma presenting as a single lesion on the nasal portion of the maxilla. Histologically, the tumor within bone and skin exhibited cords and nests of plump, epithelioid-appearing cells exhibiting rudimentary vascular differentiation within a myxohyaline stroma. Immunohistochemical reactivities for factor VIII-related antigen, CD31, CD34, and vimentin were demonstrated. Wide local excision with close clinical follow-up seems to be the treatment of choice for these tumors.