Biomedical subjects
Keith Graham
Publications and source records attributed to Keith Graham.
Prehospital pharmacology: diltiazem.
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Preclinical evaluation of the breast cancer cell-binding peptide, p160.
PURPOSE: Selective delivery of drugs into the target tissue is expected to result in high drug concentrations in the tissue of interest and therefore enhanced drug efficacy. To develop a peptide-based radiopharmaceutical, we investigated the properties of a peptide with affinity for human breast cancer, which has been selected through phage display. EXPERIMENTAL DESIGN: The bioactivity of the p160 peptide (VPWMEPAYQRFL) was evaluated in vitro and in vivo. The specific binding to human breast cancer MDA-MB-435 cells was confirmed in competition experiments. Internalization of the peptide was investigated with confocal microscopy. Furthermore, the biodistribution of (131)I-labeled p160 was studied in tumor-bearing mice. In vivo stability was evaluated at different periods after tracer administration using high-performance liquid chromatography analysis. RESULTS: The binding of (125)I-labeled p160 was inhibited up to 95% by the unlabeled peptide with an IC(50) value of 0.6 micromol/L. In addition, 40% of the total bound activity was found to be internalized into the human breast cancer cells. Although a rapid degradation was seen, biodistribution studies in nude mice showed a higher uptake in tumor than in most of the organs. Perfusion of the animals caused a reduction of the radioligand accumulation in the healthy tissues, whereas the tumor uptake remained constant. A comparison of [(131)I]p160 with a (131)I-labeled Arg-Gly-Asp peptide revealed a higher tumor-to-organ ratio for [(131)I]p160. CONCLUSIONS: p160 has properties that make it an attractive carrier for tumor imaging and the intracellular delivery of isotopes or chemotherapeutic drugs.
Pharmacological properties of hydrophilic and lipophilic derivatives of octreotate.
Derivatives of somatostatin (SST) represent the most important peptides for receptor targeting in oncological applications. Whereas the pharmacophor in somatostatin receptor-affine substances has been thoroughly investigated, the influence of modifications at the N-terminal has not yet been systematically studied. In order to investigate the influence of hydrophilic versus lipophilic modifications at the N-terminal end, a series of homologous derivatives of Tyr3-octreotate modified with oligomers of ethylene glycol or fatty acids were synthesized. For this purpose, Tyr3-octreotate was assembled using solid phase peptide synthesis and the fatty acids or oligomers of ethylene glycol were conjugated to the N-terminal end. The oligomers of ethylene glycol were activated by 4-nitrophenylchloroformate to obtain carbamate-linked hydrophilic compounds. The receptor affinities of these compounds were determined by competition experiments with [125I]Tyr3-octreotide on rat cortex membranes. The hydrophilic derivatives and the short chain lipophilic derivatives revealed IC50 values between 0.66 +/- 0.02 nM and 2.16 +/- 0.31 nM respectively. After labeling with (125)I the organ distribution of selected derivatives was investigated in Lewis rats bearing the rat pancreatic tumor CA20948. All of the compounds showed high tumor uptake. The peptides conjugated to oligomers of ethylene glycol showed low uptake into the liver and kidneys. Increasing the length of the fatty acids resulted in a remarkable decrease in kidney uptake. In conclusion, the systematic modifications at the N-terminal result in a low effect on the receptor affinity but allow the modulation of the pharmacokinetic properties of octreotide derivatives.
Fluorescent somatostatin receptor probes for the intraoperative detection of tumor tissue with long-wavelength visible light.
Targeted fluorescent dyes are of substantial value for the intraoperative delineation of primary tumors and metastatic lesions. For this purpose long-wavelength red light (lambda=550-650 nm) offers advantages because of good tissue penetration and direct visibility. Since somatostatin receptors (SSTR) are overexpressed in a number of tumors, a series of potentially tumor-selective peptide-dye conjugates were synthesized by solid-phase peptide synthesis (SPPS). The octapeptides octreotate, Tyr(3)-octreotate and Tyr(3)-octreotide were employed and exhibited high affinity for somatostatin receptors (SSTR). The fluorescent dyes rhodamine 101, sulforhodamine B acid chloride, sulforhodamine 101 or rhodamine B isothiocyanate were conjugated either directly or via spacers, for example the peptidase-labile pentapeptide sequence Ala-Leu-Ala-Leu-Ala. The conjugates were completely assembled on the solid support: Fmoc-SPPS, cyclization via a disulfide linkage, N-terminal attachment of a spacer, and linkage to the fluorescent dye. An in vitro competition assay revealed that the conjugates bind to SSTRs with IC(50) values between 0.7 and 89 nM. The conjugates were generally stable to hydrolysis at pH 7-8 in buffer or serum. However, the rhodamine 101 conjugates revealed a loss of absorption at alkaline pH due to conversion to a neutral spirolactam form, as characterized by NMR.
Eastern equine encephalitis presenting as psychosis.
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