Tracer development and PET studies : labeled proinsulin C-peptide and an EGFR-TK inhibitor

Positron emission tomography (PET), which localizes and quantifies positron decays over time, enables non-invasive in vivo distribution studies of trace amounts of compounds labeled with positron emitters. The use of PET to image disease processes is growing as the number of available tracers increases and, especially, as they become more selective and specific. PET is also increasingly being used in the development of drugs. Since trace amounts of the drug can be administered, distribution and kinetic studies can be performed in humans early in the developmental process to ascertain that the intended biophase is actually reached and to a satisfactory extent and duration.

This thesis includes (1) developments for an imaging approach with oncological application, which is based on an inhibitor of the epidermal growth factor receptor-tyrosine kinase (EGFR-TK) and (2) drug development with respect to studying the pharmacokinetics and biodistribution of proinsulin C- peptide, which is being evaluated for treating long-term complications in type 1 diabetes mellitus (DM).

The EGFR, normally involved in mitogenic signaling, is over-expressed in many different cancer types, e.g. non-small cell lung, glial cell, head and neck, pancreatic, bladder, prostate and breast cancer. A correlation between the amount of EGFR and poor prognosis has been established in a number of these cancers. A radiotracer selective for the EGFR could therefore be of interest for tumor localization and for treatment selection or evaluation of therapeutic response.

Methods to produce a precursor for radiolabeling PD153035 (4-(3-bromoanilino)- 6,7dimethoxyquinazoline), a potent inhibitor selective for the EGFR-TK (Ki~5.2 pM; 1C50~29 pM), were sought. Synthetic strategies for producing 6- or 7- mono-desmethyl and 6,7-didesmethyl PD15305 were identified. Thus different positions for labeling can be selected, which is of potential interest for metabolic studies. [6-Methoxy-11C]PD153035 was produced and its in vivo distribution to normal and proliferating tissue was evaluated in rats. In healthy rat, radioactivity was observed mainly in liver, heart, brain and in the gastrointestinal tract. Tumor radioactivity concentrations, studied in rats implanted with a human neuroblastoma cell line (SH- SY5Y), varied in accordance with differences in tumor size and viability (determined post-mortem). The results motivate further studies to determine the selectivity and specificity of [11C]PD153035 distribution to tumor in order to elucidate whether this tracer has potential for EGFR-TK level imaging with PET.

Type 1 (insulin-dependent) DM, characterized by impaired or no insulin production, is accompanied by an increased risk for micro (kidneys, nerves, retina) and macro (heart, brain, extremities) vascular dysfunction. Since insulin is lacking, so is C-peptide, which is normally produced and released in equimolar amounts together with insulin. Studies have demonstrated that administration of C-peptide to IDDM patients results in increased blood flow to skeletal muscle and skin, improved renal function and amelioration of diabetic neuropathy.

A multi-step procedure to label human C-peptide with fluorine-18 based on the activated ester N- suceinimidyl-4-[18F]fluorobenzoate ([18F]SFB) to afford N-4-[18F]fluorobenzoyl-C-peptide ([18F]FB-C-peptide) was identified. With the purpose of furthering the knowledge on Cpeptide's in vivo distribution, with special emphasis on the kinetics in the kidneys, [18F]FB-Cpeptide was studied in monkeys and in volunteering type 1 DM patients using PET. Following iv administration of [18F]FB-C-peptide, radioactivity distributed mainly to the kidneys. A tendency, albeit not statistically significant due to the small number of subjects, to slower kinetics in kidney and liver was observed when pharmacological doses of C-peptide were co-administered with the tracer. Low concentrations of radioactivity in skeletal muscle could, due to their large total mass in the body, account for approximately 15% of the administered dose. The distribution findings are consistent with the documented beneficial effects engendered by C-peptide on renal function and on blood flow to skeletal muscle.

List of scientific papers

I. Synthesis of [methoxy-11C]PD153035, a selective EGF receptor tyrosine kinase inhibitor. (1998). "Johnstrom P, Fredriksson A, Thorell JO, Stone-Elander S" J Labelled Cpd. Radiopharm 41: 623-9

II. Fredriksson A, Johnstrom P, Thorell JO, von Heijne G, Hassan M, Eksborg S, Kogner P, Borgstrom P, Ingvar M, Stone-Elander S (1999). "In vivo evaluation of the biodistribution of 11C-labeled PD153035 in rats without and with neuroblastoma implants. " Life Sci 65(2): 165-74
https://pubmed.ncbi.nlm.nih.gov/10416822

III. Fredriksson A, Stone-Elander S (2002). "Rapid microwave-assisted cleavage of methyl phenyl ethers: New method for synthesizing desmethyl precursors and for removing protecting groups." J Labelled Cpd. Radiopharm 45: 529-38

IV. Fredriksson A, Johnstrom P, Stone-Elander S, Jonasson P, Nygren PA, Ekberg K, Johansson BL, Wahren J (2001). "Labelling of human C-peptide by conjugation with N-succinimidyl-4-[18F]fluorobenzoate." J Labelled Cpd. Radiopharm 44: 509-19

V. Fredriksson A, Ekberg K, Ingvar M, Johansson BL, Wahren J, Stone-Elander S (2002). "In vivo biodistribution and pharmacokinetics of 18F-labeled human C-peptide: Evaluation in monkeys using positron emission tomography, PET." Life Sci (In Print)

VI. Fredriksson A, Ekberg K, Ingvar M, Johansson BL, Wahren J, Stone-Elder S (2002). "In vivo biodistribution of 18F-labeled C-peptide in humans: Evaluation using PET." (Manuscript)

VII. Fredriksson A, Thorell JO, Stone-Elander S (supporting data) (2002). "App. till artikel 3: Synthesis of 4-(3-bromoanilino)-7-hydroxy-6-methoxyquinazoline, 7 (7-des-methylPD150335): Preparation of a reference compound."