Barbara Stolz

Somatostatin analogs for diagnosis and treatment of cancer

Somatostatin (SRIF) is a cyclic tetradecapeptide hormone initially isolated from ovine hypothalami. It inhibits endocrine and exocrine secretion, as well as tumor cell growth, by binding to specific cell surface receptors. Its potent inhibitory activity, however, is limited by its rapid enzymatic degradation and the consequent short plasma half-life. Octreotide is a short SRIF analog with increased duration of action compared to SRIF. Octreotide is approved for the treatment of acromegaly, amine precursor uptake and decarboxylation-omas, complications of pancreatic surgery and severe forms of diarrhea. Preclinical studies have focussed on the anticancer effects of octreotide and the related SRIF analogs BIM 23014 and RC-160. In vitro at nanomolar concentrations, these analogs inhibit the growth of tumor cells that express high affinity SRIF receptors. Accordingly, SRIF analogs, such as octreotide, potently inhibit the growth of SRIF receptor-positive tumors in various rodent models, and, in particular, xenotransplanted human tumors in nude mice. The range of cancers susceptible to octreotide and related SRIF analogs includes mammary, pancreatic, colorectal and lung malignancies. Moreover, an indirect antiproliferative effect of SRIF analogs is achievable in SRIF receptor-negative tumors, whose growth is driven by factors (gastrin, insulin-like growth factor-1, etc.) that are downregulated by SRIF. The use of radiolabeled somatostatin analogs represents a new diagnostic approach. [111In-DTPA]octreotide was developed for gamma camera imaging of SRIF receptor-positive malignancies, such as gasteroenteropancreatic tumors. Visualization of SRIF receptor-positive tumors in humans is emerging as an important methodology, both in tumor staging and predicting therapeutic response to octreotide. Recently, five SRIF receptor subtypes (SSTR1-5) have been cloned, all of which bind SRIF with high affinity. In contrast, SRIF receptor subtypes 1-5 have different binding profiles for short SRIF analogs. Octreotide, SSTR5, show moderate affinity for SSTR3 and fail to bind with high affinity to the other subtypes (SSTR1 and 4). Accordingly, the oncological profile of these three analogs is apparently similar. In conclusion, somatostatin analogs are a promising class of compounds for diagnosis and treatment of cancer. Current work is focussed on the identification of further SRIF receptor subtype-selective analogs with potential in oncology.

Drug design at peptide receptors

Somatostatin (SRIF, somatotropin release inhibiting factor), discovered for its inhibitory action on growth hormone (GH) secretion from pituitary, is an abundant neuropeptide. Two forms, SRIF14 and SRIF28 exist. Recently, a second family of peptides with very similar sequences and features was described; the cortistatins (CST), CST17 and CST29 which are brain selective. The five cloned SRIF receptors (sst1–5) belong to the G-protein coupled/heptathelical receptor family. Structural and operational features distinguish two classes of receptors; SRIF1-sst2/sst3/sst5 (high affinity for octreotide or seglitide) and SRIF2=sst1/sst4 (very low affinity for the aforementioned ligands). The affinity of SRIF receptors for somatostatins and cortistatins is equally high, and it is not clear whether selective receptors do exist for one or the other of the peptides. Several radiologlands label all SRIF receptors, e.g., [125I]LTT-SRIF28, [125I]CGP23996, [125I]Tyr10cortistatin or [125I]Tyr11SRIF14. In contrast, [125I]Tyr3octreotide, [125I]BIM23027, [125I]MK678 or [125I]D-Trp8SRIF14 label predominantly SRIF1 sites, especially sst2 and possibly sst5 receptors. In brain, [125I]Tyr3octreotide binding equates with sst2 receptor mRNA distribution. Native SRIF2 receptors can be labeled with [125I]SRIF14 in the presence of high NaCl in brain (sst1) or lung (sst4) tissue. Short cyclic or linear peptide analogs show selectivity for sst2/sst5 (octreotide, lanreotide, BIM 23027), sst1 (CH-275), sst3 (sst3-ODN-8), or sst5 receptors (BIM 23268); although claims for selectivity have not always been confirmed. Beta peptides with affinity for SRIF receptors are also reported. The general lack of SRIF receptor antagonists is unique for peptide receptors, although CYN 154806 is a selective and potent sst2 antagonist. Nonpeptide ligands are still rare, although a number of molecules have been reported with selectivity and potency for sst1 (L 757,519), sst2 (L 779,976), sst3 (L 796,778), sst4 (NNC 26-9100, L 803,087) or sst1/sst5 receptors (L 817,018). Such molecules are essential to establish the role of SRIF receptors, e.g., sst1 in hypothalamic glutamate currents: sst2 in inhibiting release of GH, glucagon, TSH, gastric acid secretion, pain, seizures and tumor growth, and sst5 in vascular remodeling and inhibition of insulin and GH release.

Synthesis, radiochemistry and biological evaluation of a new somatostatin analogue (SDZ 219-387) labelled with technetium-99m

A new derivative of octreotide SDZ 219-387 [PnAO-(D)Phe1-octreotide] was synthesized, which binds specifically and with high affinity to somatostatin receptors in vitro (pK= 9.79±0.16). This new somatostatin analogue chelates technetium-99m under mild labelling conditions in good yields. The resulting [99mTc]SDZ 219–387 was stable up to 6 h after labelling and could be isolated in a pure radiochemical and chemical form by high-performance liquid chromatographic purification. The intravenous administration of purified [99mTc]SDZ 219–387 revealed that the radioligand was rapidly cleared from circulation, and tumour uptake of 0.38% ID/g was observed at 1.5 h post injection. [99mTc]SDZ 219–387 specifically interacted with somatostatin binding sites on the tumour. However, the radioligand is highly lipophilic and excreted mainly through the hepatobiliary system. As a consequence, [99mTc]SDZ 219–387 exhibits increased background activity and therefore is not appropriate for the in vivo visualization of somatostatin receptor-positive tumours and/or their metastases in the abdomen.

PET-pharmacokinetics of 18F-octreotide: A comparison with 67Ga-DFO and 86Y-DTPA-octreotide

The quantitative uptake kinetics of (2-[18F]fluoropropionyl-(D)phe1)-octreotide (I), a somatostatin (SRIF) receptor-specific tracer, was measured by PET. Conventional organ biodistribution and in vivo stabilities of the tracer as well as in vivo displacement and SRIF receptor blocking were determined. The 18F-fluorinated octreotide was compared with ([67Ga]-DFO-B-succinyl-(D)phe1)-octreotide (II) and ([86Y]-DTPA-(D)phe1)-octreotide (III). Initially, 2-10 MBq of the labeled tracers were injected into male Lewis rats bearing an exocrine pancreatic islet cell tumor. PET measurements were performed dynamically between 0 and 120 min postinjection. Organ distributions were determined 5, 15, 30, 60, and 120 min postinjection. The extent of metabolic degradation was analyzed in serial blood and urine samples as well as in homogenized samples of tumor, liver, and kidney. The uptake of (I) by the tumor was rapid (maximum accumulation at 1-2 min postinjection) and high (about 0.5 +/- 0.2% ID/g), followed by a fast and continuous release with koff = 10 +/- 2. 10(-5) s-1. The tracer was found to remain intact in vivo up to 120 min postinjection. Specific binding of (I) to SRIF receptors in the adrenals, the pancreas, and the pituitary gland was demonstrated in vivo by pretreatment and displacement experiments. Compound (II) also showed a fast uptake by the tumor. Its tumor residence half-life was longer (koff = 3.0 +/- 0.5 . 10(-5) s-1). Compound (II) was also predominantly excreted intact. One hour postinjection, the remaining activity in the blood pool was found to be bound to serum proteins. Early uptake kinetics for compound (III) were also rapid but reached only half the tumor uptake of (II). Compared to (I), the release of 86Y-activity from the tumor was slower (koff = 3.1 +/- 1.3 . 10(-5) s-1). Compared to (II), compound (III) was considerably less stable in vivo. The main critical organs for (II) and (III) are kidneys and bones, whereas (I) is predominantly accumulated in the liver. The in vivo behavior of (I) closely resembles 14C-labeled octreotide. Thus, 18F-labeled octreotide may be of interest in the quantitation and investigation of in vivo properties of somatostatin receptors by PET. However, the short residence of (2-[18F]fluoropropionyl-(D)phe1)-octreotide in tumors and its hepatobiliary excretion may complicate the interpretation of abdominal tumors.

Synthesis and Characterisation of [90Y]-Bz-DTPA-oct: A Yttrium-90-Labelled Octreotide Analogue for Radiotherapy of Somatostatin Receptor-Positive Tumours

An investigation into the in vitro behaviour of two yttrium-90-labelled somatostatin analogues was performed. Further in vivo characterisation was performed with the most promising agent. A new DTPA-octreotide analogue (Bz-DTPA-oct) was synthesised by coupling a bifunctional DTPA chelator to the N-terminal amine of the D-Phe1 of Tyr3-octreotide. This new SRIF analogue and DTPA-octreotide (OctreoScan) were radiolabelled with 90Y prior to serum stability being evaluated. Receptor binding assays were also performed on the two radioligands using rat cortex membranes. The [90Y]-Bz-DTPA-oct was further evaluated in vivo using tumour-bearing rats. The first conjugate (DTPA-octreotide) bound with a high affinity to SRIF receptors and the 90Y complex was relatively stable in human serum (t1/2 3.8 d for 90Y lost to serum proteins). The second conjugate (Bz-DTPA-oct) also exhibited a high binding affinity to SRIF receptors, but it demonstrated an even slower loss of 90Y to serum proteins (t1/2 12.1 d). The in vivo evaluation of the more stable [90Y]-Bz-DTPA-oct showed a very rapid and high accumulation in somatostatin receptor-positive tumours, which after 1 h resulted in tumour/nontumour ratios of 3.8, 21, and 4.9 (for blood, muscle, and liver, respectively). These tumour/nontumour ratios increased, and were by 24 h postinjection 138, 285, and 6.1 (for blood, muscle, and liver). Yttrium-90-labelled Bz-DTPa-oct is rapidly and selectively accumulated in somatostatin receptor-positive tissue. Octadentate Bz-DTPA-oct could be ligand for 90Y radiotherapy of somatostatin receptor-positive tumours and their metastases.

Synthesis, biodistribution and renal handling of various chelate-somatostatin conjugates with metabolizable linking groups

A series of DTPA-octreotide conjugates, with various linking groups, were synthesised to investigate the effect of different metabolizable linkers on the renal retention of radioactivity. All these newly synthesised octreotide conjugates retained the high binding affinity of octreotide for the somatostatin (SRIF) receptors either when unlabeled or radiolabeled with 111In. Some of the metabolizable linkers were rapidly degraded in vitro when incubated with a kidney homogenate. However, in vivo, all these conjugates displayed a significantly lower uptake in SRIF receptor-positive tissue compared to two conjugates with short, stable linkers. Additionally, the compounds with a potentially metabolizable linker had a higher whole-body retention of activity as opposed to the three metabolically stable compounds. Several of the linkers gave evidence of cleavage while in circulation in the blood, and it is probable that the lower tumour accumulation of most of the compounds tested was low due to the high enzymatic nature of the exocrine pancreatic tumour model used. In short, no increase in the tumour-to-kidney ratio was achieved with the analogues containing a metabolizable linker. The highest target-to-nontarget tissue ratios were obtained for the DTPA-octreotide conjugates that had short, metabolically stable linkers.

Discovery and Development of Somatostatin Agonists

Somatostatin was discovered in the laboratories of Professor R. Guillemin at the Salk Institute in La Jolla, California (Brazeau et al., 1973; Guillemin, 1992), and was first described as hypothalamic growth hormone (GH)-release inhibiting factor. Within a few years, more and more information accumulated about its ubiquitous distribution in different regions of the body, including the pancreas and gastrointestinal tract, and on its more general inhibitory functions on hormones such as insulin, glucagon, gastrin, and other gastrointestinal hormones, as well as on enzymes such as those from the exocrine pancreas. These characteristics suggested that somatostatin had enormous therapeutic potential, and early clinical investigations substantiated hopes for applications in the treatment of hypersecretory states