MEK1/2 Inhibition in Murine Heart and Aorta After Oral Administration of Refametinib Supplemented Drinking Water.

Upregulation of the RAS-RAF-MEK-ERK-MAPK pathway is involved in the development of several human tumors, aortic aneurysms, atherosclerosis, and cardiomyopathy. Refametinib, a highly selective MEK-inhibitor, has already shown antineoplastic activity in phase II trials. Furthermore, it showed potency to attenuate aortic root growth in murine models. Current formulations of this drug however necessitate oral gavage as a delivery method for long-term studies, which is labor-intensive and induces stress and occasional injury, potentially confounding results. Therefore, we developed a novel oral administration method for refametinib. A 2-hydroxypropyl-beta-cyclodextrin (HPBCD) based drinking water preparation of refametinib was formulated, for which a selective, analytical UHPLC-UV method was developed to assess the in-use stability. Next, 16 week old male wild-type C57Bl/6J mice received either a daily dose of 50 or 75 mg/kg/day refametinib or were given regular drinking water during 7 days. In both dosage groups the refametinib plasma levels were measured (n = 10 or 7, respectively). Furthermore, pERK/total ERK protein levels were calculated in the myocardial and aortic tissue of mice receiving a daily dose of 50 mg/kg/day refametinib and untreated mice (n = 4/group). After 7 days no significant degradation of refametinib was observed when dissolved in drinking water provided that drinking bottles were protected from UV/visible light. Furthermore, a dose-dependent increase in refametinib plasma levels was found whereby active plasma levels (> 1.2 µg/mL) were obtained even in the lowest dose-group of 50 mg/kg/day. A significant reduction of pERK/total ERK protein levels compared to untreated mice was observed in aortic and myocardial tissue of mice receiving a daily dose of 50 mg/kg/day refametinib. Importantly, a relatively high mortality rate was noted in the highest dose group (n = 5). This approach provides a valid alternative oral administration method for refametinib with a reduced risk of complications due to animal manipulation and without loss of functionality, which can be implemented in future research regarding the malignant upregulation of the RAS-RAF-MEK-ERK-MAPK pathway. However, care must be taken not to exceed the toxic dose.

Blood-brain barrier transport kinetics of NOTA-modified proteins: the somatropin case.

BACKGROUND: Chemical modifications such as PEG, polyamine and radiolabeling on proteins can alter their pharmacokinetic behavior and their blood-brain barrier (BBB) transport characteristics. NOTA, i.e. 1,4,7-triazacyclononane-1,4,7-triacetic acid, is a bifunctional chelating agent that has attracted the interest of the scientific community for its high complexation constant with metals like gallium. Until now, the comparative BBB transport characteristics of NOTA-modified proteins versus unmodified proteins are not yet described. METHODS: Somatropin (i.e. recombinant human growth hormone), NOTA-conjugated somatropin and gallium-labelled NOTA-conjugated somatropin were investigated for their brain penetration characteristics (multiple time regression and capillary depletion [CD]) in an in vivo mice model to determine the blood-brain transfer properties. RESULTS: The three compounds showed comparable initial brain influx, with Kin=0.38±0.14 µL/(g×min), 0.36±0.16 µL/(g×min) and 0.28±0.18 µL/(g×min), respectively. CD indicated that more than 80% of the influxed compounds reached the brain parenchyma. All three compounds were in vivo stable in serum and brain during the time frame of the experiments. CONCLUSIONS: Our results show that modification of NOTA as well as gallium chelation onto proteins, in casu somatropin, does not lead to a significantly changed pharmacokinetic profile at the blood-brain barrier.

LC-MS Compatible Antiadsorption Diluent for Peptide Analysis.

Analytical method development for peptides often proves challenging since these molecules can adsorb to the plastic or glass consumables used in the analysis. This adsorption causes considerable loss and unreliable results, especially in the lower concentration range. Therefore, a variety of antiadsorption strategies have previously been developed to cope with this adsorption, often however incompatible with direct liquid chromatography-mass spectrometry (LC-MS) analysis. Here, a novel antiadsorption diluent is introduced, based on controlled hydrolysis and precipitation of bovine serum albumin. This diluent considerably decreases the adsorption of certain peptides to glass. Moreover, it is LC-MS compatible and can also be used in combination with formic acid and/or acetonitrile addition.

In Vitro Functional Quality Characterization of NOTA-Modified Somatropins.

Chemical modifications on protein biopharmaceuticals introduce extra variability in addition to their inherent complexity, hence require more comprehensive analytical and functional characterization during their discovery, development, and manufacturing. Somatropin (i.e., recombinant human growth hormone, rhGH) modified with the chelating agent S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) allows the incorporation of radiometals for research and possible theranostic purposes. We previously demonstrated that this conjugation leads to multiple substitution degrees and positional isomers within the product. In vitro techniques at the molecular and cellular levels were now applied to assess their functional quality: (i) size exclusion chromatography (SEC) demonstrated functional complexation with human growth hormone binding protein (hGHBp) to the different NOTA-modified somatropins as well as to gallium chelated NOTA-functionalities (Ga-10:1 NOTA-somatropin); (ii) native mass spectrometry (MS) offered in-depth information, a substitution degree up to four NOTAs was still functional; (iii) circular dichroism (CD) analysis confirmed the complexation of unmodified and NOTA-modified somatropin to hGHBp; and (iv) a hGHR bioassay demonstrated initiation of the signal transduction cascade, after binding of all investigated products to the receptor presented on cells with a similar potency (pEC50 values between 9.53 and 9.78) and efficacy (Emax values between 130 and 160%). We conclude that the NOTA-modified somatropins do not possess a significantly different in vitro functionality profile compared to unmodified somatropin. Techniques such as SEC, MS, and CD, traditionally used in the physicochemical characterization of proteins have a demonstrated potential use in the functionality evaluation not only in drug discovery and development but also in quality control settings.

Hydrophilic interaction liquid chromatography method development and validation for the assay of HEPES zwitterionic buffer.

HEPES is a zwitterionic buffer component used as a raw material in the GMP-manufacturing of advanced therapy medicinal products (ATMPs), hence requiring an adequate assay method with sufficient selectivity toward related impurities. Therefore, a hydrophilic interaction chromatography (HILIC) method was developed. Different factors were investigated towards the retention behavior of HEPES, its analogue EPPS and its starting material isethionate: pH, ion concentration and organic solvent ratio of the mobile phase, as well as column temperature. Moreover, stress testing resulted in the N-oxide degradant, identified by high resolution MS. The final method consisted of an isocratic system with an aqueous (pH 2.0 with H3PO4) acetonitrile (35:65, v/v) mobile phase on a zwitterionic HILIC (Obelisc N) column with a flow rate of 0.5mL/min and UV detection at 195nm. The assay method of HEPES was validated, obtaining adequate linearity (R2=0.999), precision (RSD of 0.5%) and accuracy (recovery of 100.08%). Finally, the applicability of the validated method was demonstrated by analysis of samples from different suppliers.

Quantitative In Vitro and In Vivo Evaluation of Intestinal and Blood-Brain Barrier Transport Kinetics of the Plant N-Alkylamide Pellitorine.

Objective. To evaluate the gut mucosa and blood-brain barrier (BBB) pharmacokinetic permeability properties of the plant N-alkylamide pellitorine. Methods. Pure pellitorine and an Anacyclus pyrethrum extract were used to investigate the permeation of pellitorine through (1) a Caco-2 cell monolayer, (2) the rat gut after oral administration, and (3) the BBB in mice after intravenous and intracerebroventricular administration. A validated bioanalytical UPLC-MS(2) method was used to quantify pellitorine. Results. Pellitorine was able to cross the Caco-2 cell monolayer from the apical-to-basolateral and from the basolateral-to-apical side with apparent permeability coefficients between 0.6 · 10(-5) and 4.8 · 10(-5) cm/h and between 0.3 · 10(-5) and 5.8 · 10(-5) cm/h, respectively. In rats, a serum elimination rate constant of 0.3 h(-1) was obtained. Intravenous injection of pellitorine in mice resulted in a rapid and high permeation of pellitorine through the BBB with a unidirectional influx rate constant of 153 µL/(g·min). In particular, 97% of pellitorine reached the brain tissue, while only 3% remained in the brain capillaries. An efflux transfer constant of 0.05 min(-1) was obtained. Conclusion. Pellitorine shows a good gut permeation and rapidly permeates the BBB once in the blood, indicating a possible role in the treatment of central nervous system diseases.

Pharmaceutical Regulatory Framework in Ethiopia: A Critical Evaluation of Its Legal Basis and Implementation.

BACKGROUND: Effective and enforceable national regulations describing the manufacture and (re)packaging, export and import, distribution and storage, supply and sale, information and pharmaco-vigilance of medicines are required to consistently ensure optimal patient benefit. Expansion of pharmaceutical industries in many countries with advancement in transport technologies facilitated not only trade of genuine pharmaceutical products but also the circulation of poor quality medicines across the globe. In Ethiopia, even though "The Pharmacists and Druggists Proclamation No 43/1942" was used to regulate both the professions and the facilities where they were practiced, comprehensive regulation of the pharmaceutical market was introduced in 1964 by a regulation called "Pharmacy Regulation No. 288/ 1964". This legislation formed the legal basis for official establishment of drug regulation in the history of Ethiopia, enabling the regulation of the practice of pharmacists, druggists and pharmacy technicians; manufacturing, distribution, and sale of medicines. In June 1999, a new regulation called the "Drug Administration and Control Proclamation No. 176/1999" repealed most parts of the regulation 288/1964. The law established an independent Drug Administration and Control Authority (DACA) with further mandate of setting standards of competence for licensing institutions/facilities. DACA was re-structured as Food, Medicine and Health Care Administration and Control Authority (EFMHACA) of Ethiopia by the "Proclamation No. 661/2009" in 2010 bearing additional responsibilities like regulation of food, health care personnel and settings. The mere existence of this legal framework does not guarantee complete absence of illegal, substandard and falsified products as well as illegal establishments in the pharmaceutical chain. Therefore, the objective of the research is to assess the pharmaceutical regulatory system in Ethiopia and to reveal possible reasons for deficiencies in the pharmaceutical chain. METHODS: An archival review, an in-depth interview of key informants and an institutions-based cross-sectional survey study were conducted during March to April 2013. The comprehensiveness of the pharmaceutical law to protect public health relative to three selected African countries (South Africa, Tanzania and Uganda) and European Union, and implementation was assessed. RESULTS: The study revealed that Ethiopia does have a written national drug policy upon which the Medicines Regulatory Proclamation 661/2009 is based. According to this proclamation, the Ethiopian The Food, Medicines and Healthcare Administration and Control Authority is mandated to execute the regulatory activities as per the council of ministers regulation 189/2010. The legal framework for pharmaceutical regulation of Ethiopia was founded to fulfill all the medicines regulatory functions potentially enabling to combat illegal, substandard and falsified medicines and illegal establishments. Moreover, all the key informants witnessed that the government is commited and proclamation 661/2009 is comprehensive, but they stressed the compelling need of regulatory tools for effective implementation. From the institution-based cross-sectional study, it was revealed that there exist illegal sources formedicine in the pharmaceutical market. The main reasons for their existence were regulatory factors including weak regulatory enforcement (64.5%), lack of informal market control (60.8%), weak port control (50.0%), and poor cooperation between executive bodies (39.6%); and resource constraint (27.8%), which is an institutional factor. CONCLUSIONS: From legislative point of view, the medicines regulatory framework in Ethiopia fulfils all regulatory functions required for effective medicines regulation. However, the existence of the legislation by its own is not a guarantee to prevent the existence of unauthorized/illegal medicine sources since this requires effective implementation of the legislation, which is in fact affected by the governments political commitment, resource and intergovernmental cooperation.

Mucosal and blood-brain barrier transport kinetics of the plant N-alkylamide spilanthol using in vitro and in vivo models.

BACKGROUND: N-alkylamides (NAAs) are a large group of secondary metabolites occurring in more than 25 plant families which are often used in traditional medicine. A prominent active NAA is spilanthol. The general goal was to quantitatively investigate the gut mucosa and blood-brain barrier (BBB) permeability pharmacokinetic properties of spilanthol. METHODS: Spilanthes acmella (L.) L. extracts, as well as purified spilanthol were used to investigate (1) the permeation of spilanthol through a Caco-2 cell monolayer in vitro, (2) the absorption from the intestinal lumen after oral administration to rats, and (3) the permeation through the BBB in mice after intravenous injection. Quantification of spilanthol was performed using a validated bio-analytical UPLC-MS(2) method. RESULTS: Spilanthol was able to cross the Caco-2 cell monolayer in vitro from the apical-to-basolateral side and from the basolateral-to-apical side with apparent permeability coefficients Papp between 5.2 · 10(-5) and 10.2 · 10(-5) cm/h. This in vitro permeability was confirmed by the in vivo intestinal absorption in rats after oral administration, where an elimination rate constant ke of 0.6 h(-1) was obtained. Furthermore, once present in the systemic circulation, spilanthol rapidly penetrated the blood-brain barrier: a highly significant influx of spilanthol into the brains was observed with a unidirectional influx rate constant K1 of 796 µl/(g · min). CONCLUSIONS: Spilanthol shows a high intestinal absorption from the gut into the systemic circulation, as well as a high BBB permeation rate from the blood into the brain.

Blood-brain barrier transport kinetics of the cyclic depsipeptide mycotoxins beauvericin and enniatins.

The cyclic depsipeptide mycotoxins beauvericin and enniatins are capable of reaching the systemic circulation through various routes of exposure and are hence capable of exerting central nervous system (CNS) effects, if they are able to pass the blood-brain barrier (BBB), which was the main objective of this study. Quantification of the mycotoxins was performed using an in-house developed and validated bio-analytical UHPLC-MS/MS method. Prior to the BBB experiments, the metabolic stability of the mycotoxins was evaluated in vitro in mouse serum and brain homogenate. The BBB permeation kinetics of beauvericin and enniatins were studied using an in vivo mice model, applying multiple time regression for studying the blood-to-brain influx. Additionally, capillary depletion was applied to obtain the fraction of the peptides really entering the brain parenchyma and the fraction loosely adhered to the brain capillary wall. Finally, also the brain-to-blood efflux transport kinetics was studied. Metabolic stability data indicated that the investigated mycotoxins were stable during the duration of the in vivo study. The brain influx study showed that beauvericin and enniatins are able to cross the blood-brain barrier in mice: using the Gjedde-Patlak biphasic model, it was shown that all investigated mycotoxins exert a high initial influx rate into the brain (K1 ranging from 11 to 53µL/(g×min)), rapidly reaching a plateau. After penetration, the mycotoxins reached the brain parenchyma (95%) with only a limited amount residing in the capillaries (5%). Negligible efflux (<0.005min(-1)) from the brain was observed in the 15min post-intracerebroventricular injection.

Blood-brain barrier transport kinetics of the neuromedin peptides NMU, NMN, NMB and NT.

The neuromedin peptides are peripherally and centrally produced, but until now, it is generally believed that they only function as locally acting compounds without any quantitative knowledge about their blood-brain barrier (BBB) passage. Here, we characterize the transport kinetics of four neuromedins (NMU, NMN, NMB and NT) across the BBB, as well as their metabolization profile, and evaluate if they can act as endocrine hormones. Using the in vivo mouse model, multiple time regression (MTR), capillary depletion (CD) and brain efflux studies were performed. Data was fitted using linear (NMU, NT and NMB) or biphasic modeling (NMU and NMN). Three of the four investigated peptides, i.e. NMU, NT and NMN, showed a significant influx into the brain with unidirectional influx rate constants of 1.31 and 0.75 µL/(g × min) for NMU and NT respectively and initial influx constants (K1) of 72.14 and 7.55 µL/(g × min) and net influx constants (K) of 1.28 and 1.36 × 10(-16) µL/(g×min) for NMU and NMN respectively. The influx of NMB was negligible. Only NMN and NT showed a significant efflux out of the brain with an efflux constant (kout) of 0.042 min(-1) and 0.053 min(-1) respectively. Our results indicate that locally produced neuromedin peptides and/or fragments can be transported through the whole body, including passing the BBB, and taken up by different organs/tissues, supporting the idea that the neuromedins could have a much bigger role in the regulation of biological processes than currently assumed.

Exploration of the Medicinal Peptide Space.

The chemical properties of peptide medicines, known as the 'medicinal peptide space' is considered a multi-dimensional subset of the global peptide space, where each dimension represents a chemical descriptor. These descriptors can be linked to biofunctional, medicinal properties to varying degrees. Knowledge of this space can increase the efficiency of the peptide-drug discovery and development process, as well as advance our understanding and classification of peptide medicines. For 245 peptide drugs, already available on the market or in clinical development, multivariate dataexploration was performed using peptide relevant physicochemical descriptors, their specific peptidedrug target and their clinical use. Our retrospective analysis indicates that clusters in the medicinal peptide space are located in a relatively narrow range of the physicochemical space: dense and empty regions were found, which can be explored for the discovery of novel peptide drugs.

Regulatory development of geriatric medicines: To GIP or not to GIP?

Geriatric patients represent the main users of medicines, but are historically often minimally included in clinical trials, resulting in a gap in the knowledge of the benefit/risk balance of medicines in this heterogeneous population. As the worldwide population is aging, the need for safe and effective medicines for older patients is proportionally increasing. The aim of this review is to provide an overview of the current regulatory status of the development of geriatric medicines, the encountered challenges and the view of the involved stakeholders, coming to the conclusion whether it is necessary or not to implement a Geriatric Investigation Plan (GIP), by analogy with pediatrics.

An analytical quality by design (aQbD) approach for a L-asparaginase activity method.

L-asparaginase is an effective anti-tumor agent for acute lymphoblastic leukemia. This work presents the development of an activity determination of L-ASNase preparations for pharmaceutical quality control purposes, in accordance with analytical Quality by Design principles. Critical method attributes, the absorbance at 450 nm (A450) of the Nessler product as well as its variability, were evaluated as a function of critical method variables, by using experimental designs. The design space of the enzyme activity assay was defined (Nessler method: C(KI)/C(HgI2) of 1.90-1.95, C(NaOH)/C(HgI2) of 17.0-18.0, C(HgI2final) of 20-40 mM and time of 10-40 min; enzyme activity conditions: temperature range of 36.6-37.4 °C, pH range of the KH2PO4 buffer from 7.1 to 7.7, KH2PO4 buffer concentration: 0.18-0.22 M and L-Asn concentration of 18-22 mM), leading to a final enzyme activity assay method. A control strategy was ultimately implemented using system suitability tests.

Quality control of cationic cell-penetrating peptides.

During fundamental research, it is recommended to evaluate the test compound identity and purity in order to obtain reliable study outcomes. For peptides, quality control (QC) analyses are routinely performed using reversed-phase liquid chromatography coupled to an ultraviolet (UV) detector system. These traditional QC methods, using a C18 column and a linear gradient with formic acid (FA) as acidic modifier in the mobile phase, might not result in optimal chromatographic performance for basic peptides due to their cationic nature and hence may lead to erroneous results. Therefore, the influence of the used chromatographic system on the final QC results of basic peptides was evaluated using five cationic cell-penetrating peptides and five C18-chromatographic systems, differing in the column particle size (high performance liquid chromatography (HPLC) versus ultra-high performance liquid chromatography (UHPLC)), the acidic modifier (FA versus trifluoroacetic acid (TFA)), and the column temperature (30 °C versus 60 °C). Our results indicate that a UHPLC system with the C18 column thermostated at 30 °C and a mobile phase containing TFA, provides the most suitable routine QC analysis method for cationic peptides, outperforming in sensitivity and resolution compared to the other systems. We also demonstrate the use of a single quad mass spectrometry (MS) detector system during QC analysis of (cationic) peptides, allowing identification of the peptide and its impurities, as well as the evaluation of the peak purity.

Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo.

Cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47-57, transportan 10 (TP10) and TP10-2, were determined. The results of the multiple time regression (MTR) analysis revealed that CPPs show divergent BBB influx properties: Tat 47-57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 µl/(g × min), 5.63 µl/(g × min) and 6.02 µl/(g × min), respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA) did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.

Peptide profiling of Internet-obtained Cerebrolysin using high performance liquid chromatography - electrospray ionization ion trap and ultra high performance liquid chromatography - ion mobility - quadrupole time of flight mass spectrometry.

Cerebrolysin, a parenteral peptide preparation produced by controlled digestion of porcine brain proteins, is an approved nootropic medicine in some countries. However, it is also easily and globally available on the Internet. Nevertheless, until now, its exact chemical composition was unknown. Using high performance liquid chromatography (HPLC) coupled to ion trap and ultra high performance liquid chromatography (UHPLC) coupled to quadrupole-ion mobility-time-of-flight mass spectrometry (Q-IM-TOF MS), combined with UniProt pig protein database search and PEAKS de novo sequencing, we identified 638 unique peptides in an Internet-obtained Cerebrolysin sample. The main components in this sample originate from tubulin alpha- and beta-chain, actin, and myelin basic protein. No fragments of known neurotrophic factors like glial cell-derived neurotrophic factor (GDNF), neurotrophin nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) were found, suggesting that the activities reported in the literature are likely the result of new, hitherto unknown cryptic peptides with nootropic properties.

Blood-brain transfer and antinociception of linear and cyclic N-methyl-guanidine and thiourea-enkephalins.

Enkephalins are active in regulation of nociception in the body and are key in development of new synthetic peptide analogs that target centrally located opioid receptors. In this study, we investigated the in vivo blood-brain barrier (BBB) penetration behavior and antinociceptive activity of two cyclic enkephalin analogs with a thiourea (CycS) or a N-methyl-guanidine bridge (CycNMe), and their linear counterparts (LinS and LinNMe) in mice, as well as their in vitro metabolic stability. (125)I-LinS had the highest blood-brain clearance (K1=3.46µL/gmin), followed by (125)I-LinNMe, (125)I-CycNMe, and (125)I-CycS (K1=1.64, 0.31, and 0.11µL/gmin, respectively). Also, these peptides had a high metabolic stability (t1/2>1h) in mouse serum and brain homogenate, and half-inhibition constant (Ki) values in the nanomolar range with predominantly µ-opioid receptor selectivity. The positively charged NMe-enkephalins showed a higher antinociceptive activity (LinNMe: 298% and CycNMe: 205%), expressed as molar-dose normalized area under the curve (AUC) relative to morphine, than the neutral S-enkephalins (CycS: 122% and LinS: 130%).

Related impurities in peptide medicines.

Peptides are an increasingly important group of pharmaceuticals, positioned between classic small organic molecules and larger bio-molecules such as proteins. Currently, the peptide drug market is growing twice as fast as other drug markets, illustrating the increasing clinical as well as economical impact of this medicine group. Most peptides today are manufactured by solid-phase peptide synthesis (SPPS). This review will provide a structured overview of the most commonly observed peptide-related impurities in peptide medicines, encompassing the active pharmaceutical ingredients (API or drug substance) as well as the finished drug products. Not only is control of these peptide-related impurities and degradants critical for the already approved and clinically used peptide-drugs, these impurities also possess the capability of greatly influencing initial functionality studies during early drug discovery phases, possibly resulting in erroneous conclusions. The first group of peptide-related impurities is SPPS-related: deletion and insertion of amino acids are related to inefficient Fmoc-deprotection and excess use of amino acid reagents, respectively. Fmoc-deprotection can cause racemization of amino acid residues and thus diastereomeric impurities. Inefficient deprotection of amino acid side chains results into peptide-protection adducts. Furthermore, unprotected side chains can react with a variety of reagents used in the synthesis. Oxidation of amino acid side chains and dimeric-to-oligomeric impurities were also observed. Unwanted peptide counter ions such as trifluoroacetate, originating from the SPPS itself or from additional purification treatments, may also be present in the final peptide product. Contamination of the desired peptide product by other unrelated peptides was also seen, pointing out the lack of appropriate GMP. The second impurity group results from typical peptide degradation mechanisms such as ß-elimination, diketopiperazine, pyroglutamate and succinimide formation. These SPPS- and degradation-related impurity types can also found in the finished peptide drug products, which can additionally contain a third group of related impurities, i.e. the API-excipient degradation products.

Analytical characterization of NOTA-modified somatropins.

Chemical modification of biomolecules like the introduction of metal-chelators into proteins can lead to heterogeneous product formation. The nature and extend of the modification is important in interpreting the biological properties of the bioconjugate, given their possible influence on the pharmacokinetics as well as on the binding affinity to the target. The present study describes the synthesis and analytical characterization of somatropin modified on its lysine's ɛ-amino groups with the acylating chelator S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA). Direct separation and identification techniques (i.e. RP-MS and CE-MS) and peptide mapping after trypsin and chymotrypsin digestion demonstrated that the use of higher amounts of p-SCN-Bn-NOTA during synthesis leads to a complex product composition with higher order substitution degrees (i.e. multiple NOTA-moieties per somatropin molecule), as well as the presence of different position isomers. From the nine lysine (Lys) residues in somatropin, Lys-70 was experimentally found to be the modification hotspot under our synthesis conditions (pH=9.0). This was supported by the in silico calculated lowest pKa value of 8.3 for Lys-70. Based on the crystal structure of somatropin in complex with the extracellular parts of the growth hormone receptor, the Lys-70 residue is positioned outside the binding pockets and will therefore not directly interfere with receptor binding. Gallium chelation by NOTA-somatropin resulted in a 100% complexation. The synthesis of NOTA-somatropin using p-SCN-Bn-NOTA and somatropin under our operational conditions is therefore a suitable synthesis procedure for the production of a target-specific radiopharmaceutical for further investigation toward treatment and visualization of growth hormone-specific cancers.

Development of peptide and protein based radiopharmaceuticals.

Radiolabelled peptides and proteins have recently gained great interest as theranostics, due to their numerous and considerable advantages over small (organic) molecules. Developmental procedures of these radiolabelled biomolecules start with the radiolabelling process, greatly defined by the amino acid composition of the molecule and the radionuclide used. Depending on the radionuclide selection, radiolabelling starting materials are whether or not essential for efficient radiolabelling, resulting in direct or indirect radioiodination, radiometal-chelate coupling, indirect radiofluorination or (3)H/(14)C-labelling. Before preclinical investigations are performed, quality control analyses of the synthesized radiopharmaceutical are recommended to eliminate false positive or negative functionality results, e.g. changed receptor binding properties due to (radiolabelled) impurities. Therefore, radionuclidic, radiochemical and chemical purity are investigated, next to the general peptide attributes as described in the European and the United States Pharmacopeia. Moreover, in vitro and in vivo stability characteristics of the peptides and proteins also need to be explored, seen their strong sensitivity to proteinases and peptidases, together with radiolysis and trans-chelation phenomena of the radiopharmaceuticals. In vitro biomedical characterization of the radiolabelled peptides and proteins is performed by saturation, kinetic and competition binding assays, analyzing KD, Bmax, kon, koff and internalization properties, taking into account the chemical and metabolic stability and adsorption events inherent to peptides and proteins. In vivo biodistribution can be adapted by linker, chelate or radionuclide modifications, minimizing normal tissue (e.g. kidney and liver) radiation, and resulting in favorable dosimetry analyses. Finally, clinical trials are initiated, eventually leading to the marketing of radiolabelled peptides and proteins for PET/SPECT-imaging and therapy of different clinical diseases.