Evaluation of the therapeutic efficacy of 213Bi-labelled DOTA-conjugated alpha-melanocyte stimulating hormone peptide analogues in melanocortin-1 receptor positive preclinical melanoma model.

Melanocortin-1 receptor (MC1-R) targeting alpha-melanocyte stimulating hormone-analogue (α-MSH) biomolecules labelled with α-emitting radiometal seem to be valuable in the targeted radionuclide therapy of MC1-R positive melanoma malignum (MM). Herein is reported the anti-tumor in vivo therapeutic evaluation of MC1-R-affine [213Bi]Bi-DOTA-NAPamide and HOLDamide treatment in MC1-R positive B16-F10 melanoma tumor-bearing C57BL/6J mice. On the 6th, 8th and 10th days post tumor cell inoculation; the treated groups of mice were intravenously injected with approximately 5 MBq of both amide derivatives. Beyond body weight and tumor volume assessment, [68Ga]Ga-DOTA-HOLDamide and NAPamide-based PET/MRI scans, and ex vivo biodistribution studies were executed 30,- and 90 min postinjection. In the PET/MRI imaging studies the B16-F10 tumors were clearly visualized with both 68Ga-labelled tracers, however, significantly lower tumor-to-muscle (T/M) ratios were observed by using [68Ga]Ga-DOTA-HOLDamide. After alpha-radiotherapy treatment the tumor size of the control group was larger relative to both treated cohorts, while the smallest tumor volumes were observed in the NAPamide-treated subclass on the 10th day. Relatively higher [213Bi]Bi-DOTA-NAPamide accumulation in the B16-F10 tumors (%ID/g: 2.71 ± 0.15) with discrete background activity led to excellent T/M ratios, particularly 90 min postinjection. Overall, the therapeutic application of receptor selective [213Bi]Bi-DOTA-NAPamide seems to be feasible in MC1-R positive MM management.

In Vivo evaluation of newly synthesized 213Bi-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptide analogues in melanocortin-1 receptor (MC1-R) positive experimental melanoma model.

Given the rising pervasiveness of melanocortin-1 receptor (MC1-R) positive melanoma malignum (MM) and pertinent metastases, radiolabelled receptor-affine alpha-melanocyte stimulating hormone-analogue (α-MSH analogue) imaging probes would be of crucial importance in timely tumor diagnostic assessment. Herein we aimed at investigating the biodistribution and the MM targeting potential of newly synthesized 213Bi-conjugated MC1-R specific peptide-based radioligands with the establishment of MC1-R overexpressing MM preclinical model. DOTA-conjugated NAP, -HOLD, -FOLD, -and MARSamide were labelled with 213Bi. Ex vivo biodistribution studies were conducted post-administration of 3.81 ± 0.32 MBq [213Bi]Bi-DOTA conjugated deriva-tives into twenty B16-F10 tumor-bearing C57BL/6 J and healthy mice. Organ Level Internal Dose Assessment (OLINDA) and IDAC-Dose were used to calculate translational data-based absorbed radiation dose in human organs. Moderate or low %ID/g uptake of [213Bi]Bi-DOTA conjugated NAP, -HOLD, -and MARSamide and significantly increased [213Bi]Bi-DOTA-FOLDamide accumulation was observed in the thoracic and abdominal organs (p ≤ 0.01). High [213Bi]Bi-DOTA-NAP (%ID/g:3.76 ± 0.96), -and FOLDamide (%ID/g:3.28 ± 0.95) tumor tracer activity confirmed their MC1-R-affinity. The bladder wall received the highest radiation absorbed dose followed by the kidneys (bladder wall: 1.95·10-2 and 8.97·10-2 mSv/MBq; kidneys: 7.47·10-3 vs. 5.88·10-2 mSv/MBq measured by IDAC and OLINDA; respectively) indicating the suitability of the NAPamide derivative for clinical use. These novel [213Bi]Bi-DOTA-linked peptide probes displaying meaningful MC1-R affinity could be promising molecular probes in MM imaging.

An ω-3, but Not an ω-6 Polyunsaturated Fatty Acid Decreases Membrane Dipole Potential and Stimulates Endo-Lysosomal Escape of Penetratin.

Although the largely positive intramembrane dipole potential (DP) may substantially influence the function of transmembrane proteins, its investigation is deeply hampered by the lack of measurement techniques suitable for high-throughput examination of living cells. Here, we describe a novel emission ratiometric flow cytometry method based on F66, a 3-hydroxiflavon derivative, and demonstrate that 6-ketocholestanol, cholesterol and 7-dehydrocholesterol, saturated stearic acid (SA) and ω-6 γ-linolenic acid (GLA) increase, while ω-3 α-linolenic acid (ALA) decreases the DP. These changes do not correlate with alterations in cell viability or membrane fluidity. Pretreatment with ALA counteracts, while SA or GLA enhances cholesterol-induced DP elevations. Furthermore, ALA (but not SA or GLA) increases endo-lysosomal escape of penetratin, a cell-penetrating peptide. In summary, we have developed a novel method to measure DP in large quantities of individual living cells and propose ALA as a physiological DP lowering agent facilitating cytoplasmic entry of penetratin.

Statin-boosted cellular uptake and endosomal escape of penetratin due to reduced membrane dipole potential.

BACKGROUND AND PURPOSE: Cell penetrating peptides are promising tools for delivery of cargo into cells, but factors limiting or facilitating their cellular uptake are largely unknown. We set out to study the effect of the biophysical properties of the cell membrane on the uptake of penetratin, a cell penetrating peptide. EXPERIMENTAL APPROACH: Using labelling with pH-insensitive and pH-sensitive dyes, the kinetics of cellular uptake and endo-lysosomal escape of penetratin were studied by flow cytometry. KEY RESULTS: We report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared with its total cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased endosomal escape of penetratin in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential. CONCLUSION AND IMPLICATIONS: These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.

Synthesis and Applications of Cinchona Squaramide-Modified Poly(Glycidyl Methacrylate) Microspheres as Recyclable Polymer-Grafted Enantioselective Organocatalysts.

This work presents the immobilization of cinchona squaramide organocatalysts on poly(glycidyl methacrylate) solid supports. Preparation of the well-defined monodisperse polymer microspheres was facilitated by comprehensive parameter optimization. By exploiting the reactive epoxy groups of the polymer support, three amino-functionalized cinchona derivatives were immobilized on this carrier. To explore the effect of the amino linker, these structurally varied precatalysts were synthesized by modifying the cinchona skeleton at different positions. The catalytic activities of the immobilized organocatalysts were tested in the Michael addition of pentane-2,4-dione and trans-ß-nitrostyrene with excellent yields (up to 98 %) and enantioselectivities (up to 96 % ee). Finally, the catalysts were easily recovered five times by centrifugation without loss of activity.

Encoding Information into Polyethylene Glycol Using an Alcohol-Isocyanate "Click" Reaction.

In this article, the capability of encoding information using a homologous series of monodisperse monomethoxypolyethylene glycols (mPEG), with a number of ethylene oxide units ranging from nEO = 5 to 8, and monodisperse linear aliphatic isocyanates containing a number of CH2 units from 3 to 7, is demonstrated. The "click" reaction of the two corresponding homologous series yielded 20 different isocyanate end-capped polyethylene glycol derivatives (mPEG-OCONHR) whose sodiated adduct ion's nominal m/z values spanned from 360 to 548, providing an average ca. 8 m/z unit for the storage of one-bit information. These mPEG-OCONHR oligomers were then used to encode information in binary sequences using a 384-well MALDI sample plate and employing the common dried-droplet sample preparation method capable of encoding 20 bit, i.e., 2.5 byte information in one spot, was employed. The information stored in the spots was read by MALDI-TOF MS using the m/z value of the corresponding mPEG-OCONHR oligomers. The capability of the method to store data was demonstrated by writing and reading a text file, visualizing a small picture and capturing a short audio file written in Musical Instrument Digital Interface (MIDI) sequence. Due to the very large similarities in the chemical structures of the encoding oligomers and their "easy to be ionized" property, as well as their very similar ionization efficiencies, the MALDI-TOF MS signal intensities from each compound was so strong and unambiguous that complete decoding could be performed in each case. In addition, the set of the proposed encoding oligomers can be further extended to attain higher bit "densities".

2'[(18)F]-fluoroethylrhodamine B is a promising radiotracer to measure P-glycoprotein function.

In vivo detection of the emergence of P-glycoprotein (Pgp) mediated multidrug resistance in tumors could be beneficial for patients treated with anticancer drugs. PET technique in combination with appropriate radiotracers could be the most convenient method for detection of Pgp function. Rhodamine derivatives are validated fluorescent probes for measurement of mitochondrial membrane potential and also Pgp function. The aim of this study was to investigate whether 2'[(18)F]-fluoroethylrhodamine B ((18)FRB) a halogenated rhodamine derivative previously synthesized for PET assessment of myocardial perfusion preserved its Pgp substrate character. ATPase assay as well as accumulation experiments carried out using Pgp(+) and Pgp(-) human gynecologic (A2780/A2780(AD) and KB-3-1/KB-V1) and a mouse fibroblast cell pairs (NIH 3T3 and NIH 3T3 MDR1) were applied to study the interaction of (18)FRB with Pgp. ATPase assay proved that (18)FRB is a high affinity substrate of Pgp. Pgp(-) cells accumulated the (18)FRB rapidly in accordance with its lipophilic character. Dissipation of the mitochondrial proton gradient by a proton ionophore CCCP decreased the accumulation of rhodamine 123 (R123) and (18)FRB into Pgp(-) cells. Pgp(+) cells exhibited very low R123 and (18)FRB accumulation (around 1-8% of the Pgp(-) cell lines) which was not sensitive to the mitochondrial proton gradient; rather it was increased by the Pgp inhibitor cyclosporine A (CsA). Based on the above data we conclude that (18)FRB is a high affinity Pgp substrate and consequently a potential PET tracer to detect multidrug resistant tumors as well as the function of physiological barriers expressing Pgp.

Decreased factor XIII levels in factor XIII A subunit Leu34 homozygous patients with coronary artery disease.

INTRODUCTION: The effect of factor XIII A subunit (FXIII-A) Val34Leu polymorphism on the risk of coronary artery disease (CAD) has been extensively studied. In this study we investigated how FXIII-A Val34Leu genotypes influence plasma factor XIII levels in patients with coronary sclerosis (CS) and myocardial infarction (MI) and how fibrinogen level modulates this effect. PATIENTS AND METHODS: 955 consecutive patients admitted for coronary angiography were categorized according to the presence or absence of significant CS and the history of MI. The frequency of FXIII-A Val34Leu polymorphism, fibrinogen, FXIII activity and antigen levels were determined. RESULTS AND CONCLUSIONS: CS or MI decreased FXIII levels in patients homozygous for FXIII-A Leu34 allele, but not in heterozygous or wild type patients. In the subgroup of patients with CS, but without the history of MI no significant effect was detected, which suggests that MI has a more prominent role. The specific activity of plasma FXIII was independent of FXIII-A Val34Leu genotype. FXIII and fibrinogen levels significantly correlated in CS+ and MI+ patients. In MI+ patients of Leu/Val or Leu/Leu genotypes and with fibrinogen levels in the lowest quartile, FXIII levels were lower than in the same patient groups, but with higher fibrinogen level. The low-scale continuous activation of blood coagulation in CAD patients could lead to parallel FXIII and fibrinogen consumption. As the same amount of thrombin activates more Leu34 FXIII than Val34 FXIII, increased FXIII consumption might be responsible for the decreased FXIII levels in Leu34 homozygous CAD patients.

Modulation of the risk of coronary sclerosis/myocardial infarction by the interaction between factor XIII subunit A Val34Leu polymorphism and fibrinogen concentration in the high risk Hungarian population.

INTRODUCTION: The results on the association of factor XIII (FXIII) A subunit (FXIII-A) Val34Leu polymorphism with the risk of myocardial infarction (MI) are rather inconclusive. The original paper and confirmatory reports demonstrated a protective effect of the mutation, but results demonstrating the lack of protection have also been published. Gene-gene and gene-environmental interactions have been proposed to be responsible for the opposing results. As the rate of change in fibrin clot permeability with increasing fibrinogen concentrations decreased stepwise with increasing number of Leu34 alleles it was proposed that the protection by Val34Leu polymorphism become effective only at higher fibrinogen concentrations. However, this hypothesis has not been tested on patients with coronary artery disease. PATIENTS AND METHODS: 955 consecutive patients admitted for coronary angiography were categorized according to the presence or absence of significant coronary sclerosis (CS) and according to positive or negative history of MI. The frequency of FXIII-A Val34Leu polymorphism, and a number of risk factors, including fibrinogen were determined in the patients. FXIII-A Val34Leu polymorphism was also investigated in a population control group of 1146 subjects. RESULTS: The presence of FXIII-A Leu34 allele or homozygous Leu34 genotype did not change the risk of CS or MI in the general Hungarian population. However, when patients with fibrinogen level in the upper quartile were separately investigated, the Leu34 allele provided a statistically significant protection against MI. CONCLUSIONS: Fibrinogen concentration modulates the effect of Leu34 allele on the risk of MI; its protective effect emerges at increasing fibrinogen concentration.

Method for the synthesis of multi-epitopic Streptococcus pyogenes lipopeptide vaccines using native chemical ligation.

The aim of this study was to investigate methods for the synthesis of highly pure, well-characterized analogues of the lipid core peptide (LCP) system. Difficulties synthesizing and purifying conventional LCP systems have led to the requirement for a technique to produce highly pure, LCP-based vaccines for potential use in human clinical trials. The current study describes methods for the attachment of lipophilic adjuvants onto multi-epitopic peptide vaccines. Described is the synthesis, using native chemical ligation, of a highly pure, tri-epitopic, group A streptococcal (GAS) lipopeptide vaccine candidate. Intranasal immunization of the described tri-epitopic GAS lipopeptide with the mucosal adjuvant cholera toxin B subunit induced high serum IgG antibody titers specific for each of the incorporated peptide epitopes.

High-throughput scintillation proximity assay for transglutaminase activity measurement.

Members of the transglutaminase enzyme family are involved in a broad range of biological phenomena, including haemostasis, apoptosis, semen coagulation, skin formation, and wound healing. A new and rapid method for measurement of transglutaminase activity is described in this article. The enzyme links tritium-labeled putrescine to biotinylated oligoglutamine, and the tritiated peptide is bound to a streptavidin-coated microtiter plate permanently covered by a thin layer of scintillant. Only the radioisotope incorporated into the peptide substrate is close enough to the scintillant molecules for photons to be produced. The signal generation depends on the transglutaminase activity, and it can be detected by appropriate light-measuring instrumentation without separation steps. The assay is sensitive, specific, linear at concentrations of tissue transglutaminase between 0.05 and 1.6m U/ml, and suitable for high-throughput measurements.

Crustacean cardioactive peptide (CCAP)-related molluscan peptides (M-CCAPs) are potential extrinsic modulators of the buccal feeding network in the pond snail Lymnaea stagnalis.

We combine electrophysiological and immunocytochemical analyses in the snail Lymnaea stagnalis of M-CCAP1 and M-CCAP2, two molluscan peptides with structure similar to crustacean cardioactive peptide CCAP, originally isolated from the snail Helix pomatia. Both M-CCAP peptides (M-CCAP1 and M-CCAP2, 1 microM) had an excitatory effect, depolarizing all the identified neurons of the buccal feeding network (including motoneurons: B1, B2, B4 and modulatory interneurons SO, OC: 62 neurons in 33 preparations). Additionally, in 67% of preparations, rhythmic activity (fictive feeding) was recorded with a mean rate of 7 cycles/min. No significant difference in the proportion of preparations showing fictive feeding or mean feeding rate was found between M-CCAP1 and M-CCAP2. The extrinsic feeding modulator, the serotonergic CGC neuron, responds by increase of the spontaneous activity after M-CCAP application (9 of 18 preparations). Crustacean CCAP (1 microM) evokes a slight membrane depolarization in 3 out of 8 preparations but never evokes fictive feeding. Immunostaining revealed no cell bodies in the buccal ganglia, but a dense network of CCAP immunopositive fibers arborizing in the buccal neuropil. Many of these fibers originate from a symmetrical pair of CCAP-immunoreactive cerebro-buccal interneurons, which are the most likely candidates for extrinsic modulatory interneurons in the buccal feeding network. Our data are the first results suggesting that M-CCAP-peptides exist as effective modulators in mollusc.

Toward the development of a synthetic group a streptococcal vaccine of high purity and broad protective coverage.

Using native chemical ligation, we synthesized a group A streptococcal (GAS) vaccine that contained three different GAS M protein peptide epitopes in a chemically well-characterized construct in high purity. Two of the peptide epitopes represented variable amino terminal serotype determinants, and the third represented a carboxyl terminal conserved region determinant of the GAS M protein. We also synthesized a lipid core peptide (LCP) construct containing the same three peptides. Upon immunization of mice, the non-LCP construct only elicited antibody responses to all three epitopes with the use of adjuvant. The LCP construct, however, elicited excellent antibody responses to all three epitopes without the need for any additional adjuvant or carrier. We have synthesized the LCP synthetic vaccine system with good reproducibility.

Platelets but not monocytes contribute to the plasma levels of factor XIII subunit A in patients undergoing autologous peripheral blood stem cell transplantation.

Factor XIII subunit A (FXIII A) is synthesized by megakaryocytes, and monocytes/macrophages. In addition, the liver has been reported as an extrahaematopoietic source of FXIII A. At present, the extent of contribution of either haematopoietic or extrahaematopoietic sources to the plasma FXIII A level is unknown. We studied the effect of bone marrow aplasia due to high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ASCT) on plasma FXIII A activity and concentration in 20 patients with haematological or solid tumour malignancies. A multiple linear regression model was used to assess the effect of gender, age, malignancy and treatment types, platelet and monocyte counts, abnormal liver function tests, prothrombin time, and number of platelet transfusions on FXIII activity measured in plasma before and following ASCT. Significant correlation between platelet counts and FXIII A activity in plasma was observed (r = 0.51, P = 0.0001), which remained after the adjustment for the aforementioned parameters (multiple R = 0.52, P = 0.0001). In contrast, no significant correlation between FXIII A levels and monocyte counts was observed (r = 0.19), and this lack of correlation persisted after the adjustment. These results suggest that in the ASCT model, following myeloablation, platelets but not monocytes are the haematopoietic cells that contribute significantly to plasma FXIII A levels. In addition, extra-haematopoietic sources of FXIII synthesis may also contribute to FXIII levels in plasma.