NIR-Cyanine Dye Linker: a Promising Candidate for Isochronic Fluorescence Imaging in Molecular Cancer Diagnostics and Therapy Monitoring.

Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner.

A peptide & peptide nucleic acid synthesis technology for transporter molecules and theranostics--the SPPS.

Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here.

SPPS resins impact the PNA-syntheses' improvement.

The personalized medicine, also documented as "individualized medicine", is an effective and therapeutic approach. It is designed to treat the disease of the individual patient whose precise differential gene expression profile is well known. The trend in the biomedical and biophysical research shows important consequences for the pharmaceutical drug and diagnostics research. It requires a high variability in the design and safety of target-specific pharmacologically active molecules and diagnostic components for imaging of metabolic processes. A key technology which may fulfill the highest demands during synthesis of these individual drugs and diagnostics is the solid phase synthesis which is congenial to automated manufacturing. Additionally the choice of tools like resins and reagents is pivotal to synthesize drugs and diagnostics in high quality and yields. Here we demonstrate the solid phase synthesis effects dependent on the choice of resin and of the deprotection agent.

Pharmacokinetics of 5-aminofluorescein-albumin, a novel fluorescence marker of brain tumors during surgery.

In a phase I/II study, the new fluorescence marker 5-aminofluorescein covalently bound to human serum albumin (AFL-HSA) was intravenously administered to 10 patients with brain tumor 1 to 4 days before surgery, and AFL-HSA kinetics were determined to assess the optimum timing of dye administration. AFL-HSA was determined in serum and brain tissue by size exclusion chromatography with fluorescence detection. AFL-HSA disposition was quite similar to albumin kinetics with small volumes of distribution (volume of central compartment [V(c)] = 2.4 L; volume of distribution [Vd(ß)] = 6.8 L; volume at steady state [V(ss)] = 5.9 L), a low clearance (mean Cl = 16.85 mL/h), and a long elimination half-life (mean T(1/2;ß) = 307 hours). Predicted peak concentrations in the peripheral compartment occurred at 80.9 hours, the time point of optimal fluorescence intensity observed in brain tumors during surgery.

Development of a novel polyethylene glycol-corticosteroid-conjugate with an acid-cleavable linker.

BACKGROUND: Corticosteroids like dexamethasone are often used in the treatment of inflammatory diseases. Despite efficacy, their use is limited by severe side-effects. Targeted drug-delivery to the site of inflammation would be advantageous for the patients. Macromolecules can be used for this approach, because they accumulate at sites of inflammation due to the enhanced permeability and retention effect. PURPOSE: Our aim was to develop a polymer-corticosteroid-conjugate for the treatment of inflammatory diseases. The authors covalently linked a derivative of dexamethasone to the macromolecule polyethylene glycol (PEG), using an acid-cleavable linker to achieve lysosomal drug-release. METHODS: The corticosteroid-PEG-conjugate was synthesized and characterized by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry. Cleavage experiments were performed to study the nature of products after incubation at acidic pH, and the efficacy of the conjugate was tested in two model cell lines. RESULTS: Acid hydrolysis of the novel corticosteroid-PEG-conjugate resulted in two new derivatives of dexamethasone. The conjugate was effective in both model cell lines showing lysosomal release and efficacy of the cleavage products. DISCUSSION AND CONCLUSION: The authors new corticosteroid-PEG-conjugate shows glucocorticoid activity and should be developed further to treat inflammatory diseases with reduced side-effects while retaining drug efficacy.

Native albumin for targeted drug delivery.

UNLABELLED: IMPORTANCE IN THE FIELD: Activated cells metabolize albumin to cover their increased need for amino acids and energy. In inflamed, diseased and malignant tissue, extravasation of macromolecules into the tissue is upregulated. Drug carriers such as albumin have been used to target specifically diseased and malignant cells, resulting in higher efficacy of treatment and reduced side effects. AREAS COVERED IN THIS REVIEW: Owing to its advantageous biochemical and pharmacological properties, albumin has been regarded as an interesting candidate as a drug carrier. Covalent coupling to albumin carries drugs specifically to tumors and sites of inflammation, leading to reduced side effects as long as the native structure of albumin is unchanged. In this review, the means of coupling drugs to native albumin as well as exemplary studies for the use of albumin as drug carrier are summarized and discussed. WHAT THE READER WILL GAIN: An overview of the state-of-the-art using albumin as drug carrier for specific accumulation in tumors and inflammatory cells using the advantageous properties of native albumin is given in this review. TAKE HOME MESSAGE: Native albumin is an effective drug carrier to sites of inflammation or malignancy.

Serum albumin leads to false-positive results in the XTT and the MTT assay.

Tetrazolium salts like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) that form formazans after reduction are widely used to investigate cell viability. Besides cellular enzymes, some constituents of cell media and other substances reduce tetrazolium salts, thereby interfering with these assays. We describe here that different preparations of serum albumin from bovine or human origin can lead to a concentration-dependent increase in the signals of the XTT assay; therefore leading to an overestimation of cell numbers and to an underestimation of potential cytotoxic effects of compounds to be tested. The same effect was seen in the MTT assay with human serum albumin (HSA). We demonstrate that this reductive activity cannot be inactivated by proteolytic digestion, but that it is due to the free cysteine residue in albumin, and is also observed when cysteine or glutathione (GSH) are used. Binding of N-ethylmaleimide (NEM) to the free cysteine residue leads to a decrease of the albumin interference in the XTT assay.

Methotrexate-albumin and aminopterin-albumin effectively prevent experimental acute graft-versus-host disease.

BACKGROUND: During the last several years, major progress has been made in developing targeted chemotherapy using cytotoxic drugs covalently bound to human serum albumin (HSA). We explored the activity of two antifolates methotrexate (MTX) and aminopterin (AMPT) bound to HSA in prophylaxis and treatment of experimental acute graft-versus-host disease (aGVHD). METHODS: In all, 113 female F1 hybrid BN/Lew-rats were irradiated with 8.2 Gy (n=103) or 9.9 Gy (n=10). One day after irradiation each rat received 4 x 10 bone marrow cells and 1.5 x 10 spleen T-cells from female Lew-rats. GVHD prophylaxis consisted of MTX-HSA 2 mg/kg (n=25), MTX-HSA 0.5 mg/kg (n=8), AMPT-HSA 0.65 mg/kg (n=8), MTX 0.5 mg/kg (n=17), or native HSA (n=39) given intraperitoneally (IP) on days 0, 4, 8, and 12. Treatment of aGVHD consisted of MTX-HSA 2 mg/kg (n=8) or AMPT-HSA 0.5 mg/kg (n=8) given intraperitoneally at the time of onset of aGVHD and subsequently every fourth day (a total of four doses). RESULTS: All animals receiving native HSA developed lethal aGVHD. Prophylactic treatment with MTX-HSA 2 mg/kg prevented aGVHD in 18 of 25 animals and in 6 of 8 receiving AMPT-HSA. In contrast, five out of nine rats receiving free MTX died due to aGVHD. The survival rates in the prophylactic MTX-HSA 2 mg/kg and AMPT-HSA groups were significantly higher compared to the MTX and control groups (P<0.05), while non hematologic toxicity of MTX-HSA was not detectable. AMPT-HSA at a dose of 0.65 mg/kg as well as MTX at a dose of 0.5 mg/kg were associated with temporary weight loss and lethargy. CONCLUSIONS: The albumin conjugates MTX-HSA and AMPT-HSA effectively prevent experimental aGVHD.

Albumin targeting of damaged muscle fibres in the mdx mouse can be monitored by MRI.

Increased sarcolemmal permeability has been implicated as a major pathological event in muscular dystrophies. In our study, we evaluated whether damaged muscle fibres can be specifically targeted using albumin as a carrier. We tagged human serum albumin (HSA) with Gadolinium (Gd) and systemically applied this compound (Gd-DTPA-HSA) to wildtype and dystrophin-deficient mdx mice. We performed magnetic resonance imaging before and after intravenous administration of Gd-DTPA-HSA and found localised signal enhancement only in mdx skeletal muscle. We also examined skeletal muscle after contrast enhanced magnetic resonance imaging using anti-human albumin antibodies and demonstrated intracellular accumulation of Gd-DTPA-HSA in clusters of damaged mdx muscle fibres. Comparison of magnetic resonance imaging and histological data emphasised the value of contrast agent enhanced magnetic resonance imaging for the in vivo assessment of fibre damage in muscular dystrophies. Furthermore, our data provide evidence that albumin can be used as a carrier to target covalently bound molecules to degenerating muscle fibres.

Efficacy and tolerability of an aminopterin-albumin conjugate in tumor-bearing rats.

The antifolate aminopterin (AMPT) was developed before methotrexate (MTX), but was not clinically established or generally used due its increased toxicity compared to MTX. Recently, we reported on the increased metabolism of albumin conjugates such as methotrexate-albumin (MTX-SA) in malignant tumors and the feasibility of using albumin as a carrier for drug targeting. Consequently, AMPT was covalently bound to serum albumin (AMPT-SA) at a 1:1 molar ratio. Biodistribution, tolerability and efficacy of this novel conjugate were studied in Walker-256 (W-256) carcinoma-bearing rats. As compared to native albumin, the same biodistribution and plasma clearance were found for AMPT-SA, which achieved 20.1% tumor uptake (estimated uptake per g tumor 6.4%) within 24 h after i.v. administration in rats. In a randomized study, AMPT-SA, repeatedly i.v. injected, was compared with low-molecular-weight AMPT. Depending on the molar concentration, the maximum tolerated dose (MTD) of AMPT covalently bound to SA was twice that of unbound AMPT (three repeated injections of 1.0 mg AMPT-SA/kg body weight versus three repeated injections of 0.5 mg AMPT/kg body weight; p=0.0006). Efficacy was studied at the level of the MTD and MTD/2, and demonstrated that AMPT-SA was significantly more active. At the MTD/2 in W-256 carcinoma-bearing rats, AMPT-SA achieved a 100% volume reduction and an optimal volume reduction during treatment/control (T/C) of 8.3% compared to a 53% volume reduction of AMPT and a T/C of 16.5% (p=0.032). Tumor relapses were reduced and occurred later in the AMPT-SA group (two tumor recurrences for AMPT-SA versus seven for AMPT; p=0.05). In this comparative study, the AMPT-SA conjugate showed high antitumor activity in vivo and a favorable toxicity compared to low-molecular-weight AMPT. These effects are attributed to the albumin carrier which seems to be an effective tool for selective tumor drug targeting.