Search

A growing industrial interest toward the peptide drug market fueled the need for the development of effective and cGMP compliant manufacturing methods for these complex molecules. Solid-phase strategies are considered methods of election for medium-length peptide syntheses not only on the research scale but for multigram-scale production, as well. The possibility to use microwave-assisted technology on the multigram scale, recently introduced, prompted us to evaluate the possibility to conveniently set up a safe and fully cGMP-compliant pilot process to produce eptifibatide, a generic peptide active pharmaceutical ingredient. Accordingly, we developed an optimized process on the laboratory scale (1-5 mmol), which was subsequently successfully scaled up to 70 mmol, obtaining all the information required by regulatory agencies to validate the process and qualify the pilot scale plant. The process consists of 5 steps: (1) automated microwave-assisted solid-phase synthesis of eptifibatide linear precursor; (2) cleavage from the resin with concomitant amino acid side-chains deprotection; (3) disulfide-bond formation in solution; (4) purification by flash column chromatography; (5) ion-exchange solid-phase extraction. Since the direct scale-up of a multigram-scale cGMP compliant peptide API production procedure is a challenge that requires an accurate understanding of each involved step, we initially performed a quality management risk assessment, which enabled a smooth and effective achievement of a successful final result.

We investigated several strategies, based on the use of microwave-assisted solid-phase peptide synthesis (MW-SPPS) and scalable to kilogram-scale manufacturing, for the preparation of Eptifibatide, a disulfide-bridged cyclo-heptapeptide drug approved as an antithrombotic agent. Following the very fast microwave-assisted Fmoc/tBu synthesis of the linear precursor, we explored both the solution (off-resin) and the solid-phase (on-resin) disulfide formation. In order to optimize the oxidation in solution, we focused our attention on the mild disulfide formation procedure based on the use of air, observing some drawbacks, such as the formation of unwanted oxidation byproducts, such as dimers, or the use of large volumes of an environmentally unfriendly solvent (CHCN). In order to overcome these difficulties, we studied four different on-resin strategies, with the final aim to develop a fully automated, single reactor procedure, exploring different strategies to protect the thiol side-chain functional group on the C-terminal Cys residue and to form the Eptifibatide ring. The main difference among these strategies is represented by the final cyclization mode that was obtained either by direct formation of an S-S disulfide bridge or by head to MPA on cysteine side-chain amide bond formation. In conclusion, the optimization of the latter strategy enabled us to devise an optimized scalable fully automated solid-phase microwave-assisted cGMP-ready process to prepare Eptifibatide.

Diagnostics of Multiple Sclerosis (MS) are essentially based on the gold standard magnetic resonance imaging. Few alternative simple assays are available to follow up disease activity. Considering that the disease can remain elusive for years, identification of antibodies fluctuating in biological fluids as relevant biomarkers of immune response is a challenge. In previous studies, we reported that anti-N-glucosylated (N-Glc) peptide antibodies that can be easily detected in Solid-Phase Enzyme-Linked ImmunoSorbent Assays (SP-ELISA) on MS patients&rsquo, sera preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus Influenzae. Since multivalency can be useful for diagnostic purposes to allow an efficient coating in ELISA, we report herein the development of a collection of Multiple N-glucosylated Peptide Epitopes (N-Glc MEPs) to detect anti-N-Glc antibodies in MS. To this aim, a series of N-Glc peptide antigens to be represented in the N-GlcMEPs were tested in competitive ELISA. We confirmed that the epitope recognized by antibodies shall contain at least 5-mer sequences including the fundamental N-Glc moiety. Using a 4-branched dendrimeric lysine scaffold, we selected the N-Glc MEP 24, carrying the minimal epitope Asn(Glc) anchored to a polyethylene glycol-based spacer (PEG) containing a 19-atoms chain, as an efficient multivalent probe to reveal specific and high affinity anti-N-Glc antibodies in MS.

The role of pathologic auto-antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35-55). In this scenario, we analyzed the anti-MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1-117). To assess the presence of a B-cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1-117 sequence of MOG, including MOG(35-55). For this purpose, we cloned, expressed in Escherichia coli and on-column refolded MOG(1-117), and we applied an optimized microwave-assisted solid-phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid-phase immunoenzymatic assay testing both naive and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35-55), we hypothesize the presence of both linear and conformational epitopes on MOG(35-55) sequence.

The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell-1 (HNK-1), considered the antigenic determinant of myelin-associated glycoprotein and the target of clinically relevant auto-antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure-activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK-1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09 x 10(-7) m for a commercial anti-HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients' sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto-antibodies.

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.

Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.

Celiac disease (CD) patients usually present high levels of circulating IgA antibodies directed to different antigens, in particular tissue transglutaminase (tTG), gliadin (Glia), and endomysium. A series of synthetic peptide constructs containing cross-linked tTG and Glia deamidated peptides have been synthesized. Peptides were tested in enzyme-linked immunosorbent assays against celiac disease patients' sera versus normal blood donors, and their conformational features were evaluated by molecular modeling techniques. Four peptides were recognized as epitopes by autoantibodies (IgG class) circulating in CD patients' sera before gluten-free diet. The peptide II, containing Ac-tTG(553-564)-NH2 sequence cross-linked with deamidated Ac-alpha 2-Glia(63-71)-NH2, was able to identify specific disease antibodies with a sensitivity of 50% and a specificity of 94.4%. Structural conformations of the linear fragments Ac-tTG(553-564)-NH2 and Ac-alpha 2-Glia(63-71)-NH2 and the corresponding cross-linked peptide II were calculated by molecular modeling. Results showed that cross-linking is determinant to assume conformations, which are not accessible to the linear fragments.

Multiple sclerosis (MS) is a complex disease that seems to depend on several pathophysiological processes. Because of its varied clinical presentation, natural history, and response to therapeutic interventions, MS can be considered to be a group of diseases that have not been yet characterized, thus resulting in difficult evaluation of prognosis. In the last few years, the role of autoAbs in MS has been reevaluated, and, therefore, their identification as specific biomarkers became a relevant target. In this paper, we demonstrate that an aberrant N -glucosylation is a fundamental determinant of autoAb recognition in MS. Thus, we developed CSF114(Glc), an antigenic probe accurately measuring IgM autoAbs in the sera of a patient population, as disease biomarker. The relevance of CSF114(Glc) is demonstrated by its clinical application and correlation with disease activity and prognosis. In fact, CSF114(Glc), a structure-based designed glycopeptide, is able to recognize, by ELISA, the presence of specific IgM autoAbs in the sera of a MS patient population but not in blood donors and other autoimmune conditions. AutoAbs specific for CSF114(Glc) isolated from MS patients recognized myelin and oligodendrocyte antigens by immunohistochemistry but not other nonrelevant tissues. We demonstrate that CSF114(Glc) is a reliable, specific probe in a longitudinal study of untreated MS patients. Development of IgG/IgM anti-CSF114(Glc) Abs paralleled clinical activity and brain lesions positive to MRI. Therefore, a CSF114(Glc)-based immunoassay on sera may have important prognostic value in monitoring MS disease progression guiding optimal therapeutic treatment.

The synthesis of a new biotin derivative, the (CO) reduced N-aminohexyl biotinamido derivative, designed to be serum biotinidase resistant, and its conjugation to the chelator DOTA through an amide bond at one of the four carboxymethyl chains are described. The (90)Y-labeled conjugate was able to bind avidin at different Av/conjugate molar ratios with good results. The preclinical results indicate that this new biotin-DOTA conjugate is a good candidate for pretargeted diagnosis and therapy of tumors.

Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n=53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n=82) and healthy age-matched controls (n=29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a syntheticN-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P=0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.