Mathew L. Thakur

Decorin Protein Core Inhibits in Vivo Cancer Growth and Metabolism by Hindering Epidermal Growth Factor Receptor Function and Triggering Apoptosis via Caspase-3 Activation

Decorin is not only a regulator of matrix assembly but also a key signaling molecule that modulates the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR). Decorin evokes protracted internalization of the EGFR via a caveolar-mediated endocytosis, which leads to EGFR degradation and attenuation of its signaling pathway. In this study, we tested if systemic delivery of decorin protein core would affect the biology of an orthotopic squamous carcinoma xenograft. After tumor engraftment, the animals were given intraperitoneal injections of either vehicle or decorin protein core (2.5-10 mg kg-1) every 2 days for 18-38 days. This regimen caused a significant and dose-dependent inhibition of the tumor xenograft growth, with a concurrent decrease in mitotic index and a significant increase in apoptosis. Positron emission tomography showed that the metabolic activity of the tumor xenografts was significantly reduced by decorin treatment. Decorin protein core specifically targeted the tumor cells enriched in EGFR and caused a significant down-regulation of EGFR and attenuation of its activity. In vitro studies showed that the uptake of decorin by the A431 cells was rapid and caused a protracted down-regulation of the EGFR to levels similar to those observed in the tumor xenografts. Furthermore, decorin induced apoptosis via activation of caspase-3. This could represent an additional mechanism whereby decorin might influence cell growth and survival.

Radiolabeled peptides in the diagnosis and therapy of oncological diseases

There has been an exponential growth in the development of radiolabeled peptides for diagnostic and therapeutic applications in oncology. Peptides have fast clearance, rapid tissue penetration, low antigenicity and can be produced easily and inexpensively. However, peptides have problems with in vivo catabolism, unwanted physiological effects, and chelate attachment. The approved 111In-DTPA-OctreoScan, a somatostatin receptor binder, is well established for diagnosis of neuroendocrine tumors. NeoTect, an approved, 99mTc-labeled, somatostatin-receptor-binding analogue has good specificity for lung cancer detection. The receptors for Vasoactive Intestinal Peptide, Cholecystokinin-B/gastrin, Bombesin, Epidermal Growth Factor, and Alpha Melanocyte Stimulating Hormone and the Integrin, alpha(v)beta(3), are under active investigation as targets. Octreotide and its analogues labeled with 111In, 90Y, 64Cu or 177Lu are under study for the treatment of patients with promising results.

An Antimetastatic Role for Decorin in Breast Cancer

Decorin, a member of the small leucine-rich proteoglycan gene family, down-regulates members of the ErbB receptor tyrosine kinase family and attenuates their signaling, leading to growth inhibition. We investigated the effects of decorin on the growth of ErbB2-overexpressing mammary carcinoma cells in comparison with AG879, an established ErbB2 kinase inhibitor. Cell proliferation and anchorage-independent growth assays showed that decorin was a potent inhibitor of breast cancer cell growth and a pro-apoptotic agent. When decorin and AG879 were used in combination, the inhibitory effect was synergistic in proliferation assays but only additive in both colony formation and apoptosis assays. Active recombinant human decorin protein core, AG879, or a combination of both was administered systemically to mice bearing orthotopic mammary carcinoma xenografts. Primary tumor growth and metabolism were reduced by approximately 50% by both decorin and AG879. However, no synergism was observed in vivo. Decorin specifically targeted the tumor cells and caused a significant reduction of ErbB2 levels in the tumor xenografts. Most importantly, systemic delivery of decorin prevented metastatic spreading to the lungs, as detected by novel species-specific DNA detection and quantitative assays. In contrast, AG879 failed to have any effect. Our data support a role for decorin as a powerful and effective therapeutic agent against breast cancer due to its inhibition of both primary tumor growth and metastatic spreading.

INDIUM-111-LABELLED LEUCOCYTES FOR LOCALISATION OF ABSCESSES

Leucocytes from eight patients who were thought to have an abscess were labelled with indium-111 and reintroduced into the circulation. The distribution of radioactivity was followed by whole-body scanning and imaging with a gamma camera. Focal accumulation of radioactivity were observed in the lesion in the three patients with abscesses, in the lungs of a boy with bacterial endocarditis, in the knee of a woman with rheumatoid arthritis, and at the site of intramuscular injections in another patient. The use of radiolabelled cells for the detection of focal pathological processes would seem to be an important addition to conventional diagnostic methods.

Radiolabeled Peptides in Oncology

There has been an exponential growth in the development of radiolabeled peptides for diagnostic and therapeutic applications in the last decade. The automated means of synthesizing these compounds in large quantities and the simplified methods of purifying, characterizing, and optimizing them have kindled attention to peptides as carrier molecules. These new techniques have accelerated the commercial development of radiolabelled peptides, which has provided additional radiopharmaceuticals for the nuclear medicine community.Peptides have many key properties including fast clearance, rapid tissue penetration, and low antigenicity, and can be produced easily and inexpensively. However, there may be problems with in vivo catabolism, unwanted physiologic effects, and chelate attachment. Radiolabeled peptides have made their greatest impact in the management of relatively rare neuroendocrine malignancies. Indeed, Indium-111 (111In)-pentetreotide (111In-DTPA-octreotide, Octreoscan®), which binds to somatostatin receptors (SSTRs), has become the diagnostic ‘gold standard’ in these diseases. However, 111In-pentetreotide has been less successful in the diagnosis of other more prevalent diseases in which SSTRs are upregulated. Technetium-99m (99mTc)-depreotide (NeoTect™), a 99mTc-labeled SSTR-analog, could have wider impact since it has high sensitivity and specificity for lung cancer lesion detection. However, this impact may be minimized by the increased availability of positron emission tomography imaging with Fluorine-18 (18F)-flourodeoxyglucose, which has similar sensitivity and specificity for lesion identification in this disease, and is currently more widely used. The receptors for bombesin, α-melanocyte-stimulating hormone, neurotensin, and the integrin αvβ3, are under active investigation as targets for radiolabelled peptides, but are still in the pre-clinical stage. Compounds directed at the cholecystokinin-B/gastrin receptor have shown promising results in clinical trials in humans.Radiolabelled peptide therapy is usually indicated for patients with widespread disease that is not amenable to focused radiation therapy or is refractory to chemotherapy. Phase I/II studies using various radiolabelled peptides (including 111In-pentetreotide, Yttrium-90 [90Y]-DOTA-Phe1-Tyr3-octreotide, 90Y-DOTA-lanreotide, and Lutetium-177 [177Lu]-DOTA-octreotate) for the treatment of patients with neuroendocrine malignancy are in progress. Over 400 patients have been treated, and the response rate has ranged from 60% to 75%, although few patients have had a complete response. Patients have been given individual doses ranging from 2 to 11 GBq with a slow infusion every 4–8 weeks (up to 12 times). The kidney is the dose-limiting organ and most patients experience a transient decline in blood cell counts. A concomitant infusion of an amino acid mixture can reduce kidney toxicity and increase the effective tumor dose. Other peptides currently under investigation, some of which have shown promising results, include Rhenium-188 (188Re)-P2045 and 90Y-αvβ3 antagonist.

Gallium-67 and indium-111 radiopharmaceuticals

Abstract There are no compounds known to exist naturally in the human body which involve gallium or indium metal cofactors. In spite of this numerous compounds which contain 67Ga and 111In as their integral part have been prepared mainly for localization studies. One of the important considerations in these preparations has been the physical characteristics of 67Ga and 111In which are summarized together with the methods of their preparation. Factors governing the chelation of organometallic radiopharmaceuticals, their in vivo stability and usefulness have been discussed. The compounds prepared have been reviewed and summarized together with the basic concept in their preparation. The results of their in vivo evaluations have been outlined and the merits for potential future applications of some of the compounds have been mentioned.

A consensus protocol for white blood cells labelling with technetium-99m hexamethylpropylene amine oxime

Since its introduction in 1976 as an imaging procedure, the use of labelled leucocyte scintigraphy has continued to increase. Nowadays it is a routine procedure in most nuclear medicine departments for the investigation of different inflammatory pathologies involving leucocytic infiltration. These include bone and soft tissue infections, inflammatory bowel disease, vascular prosthesis infection and fever of unknown origin [1–4]. Despite their non-specificity, the use of non-polar, lipid-soluble chelates is still the most efficient way to label leucocytes. Indium-111 oxine was the first to be described in 1976 [5] and has been the agent of choice for many years. Other indium chelates such as 111In-tropolone [6] and 111In-mercapto-pyridineN-oxide [7] have been demonstrated to label leucocytes as efficiently as 111In-oxine in plasma medium, but their use has not been so widespread. Although efficient in many ways, the use of 111In-oxine has become less attractive due to its cost, inconvenient supply, and the unfavourable physical characteristics of 111In. Technetium-99m has excellent characteristics for scintigraphic imaging and is cheap and available at any time. For this reason, many investigators have tried to find a 99mTc-chelate able, like 111In-chelates, to enter cells and to bind a cytoplasmatic component without loss of leucocyte viability. 99mTc-propylene amine oximes (PnAO) were originally developed as lipophilic diffusible radiopharmaceuticals for brain imaging [8]. The dl diastereoisomer complex of 99mTc-hexamethylpropylene amine oxime (HMPAO) was found to have the highest brain concentration [9]. Because of its neutral, lipophilic character, this radiopharmaceutical is able to diffuse through the cell membrane and bind intracellular components. Soon 99mTc-HMPAO proved to be useful as a leucocyte labelling agent [10, 11]. Since 1988 99mTc-HMPAO has been commercially available. Nevertheless, its use is accompanied by a large variety of labelling methodologies which, although they in general do not induce any significant damage on labelled cells, can influence the image quality and interpretation of results. The International Society of Radiolabelled Blood Elements (ISORBE), founded in 1989 in Vienna (Austria) with the primary objective of providing a forum for exchange of knowledge and experience, decided to crutinize various methods and to suggest a uniform procedure for radiolabelling leucocytes with 99mTcHMPAO.

Radiolabeled somatostatin analogs in prostate cancer

Vapreotide (RC-160), a somatostatin analog, was labeled with 99mTC by a direct method and also by using CPTA [1,4,8,11-tetraazacyclotetradecane] as a bifunctional chelating agent. The labeled compounds were evaluated in nude mice bearing experimental human prostate cancers. In these studies, 111In-DTPA-D-Phe-Octreotide (111In-DTPA-octreotide) served as a standard and 99mTc-oxytocin as a receptor-non-specific control. 99mTc-octreotide was also used. The 24 htumor uptake of 99mTc-RC-160 was nearly 400% higher, (p < 0.05), than that of 111In-DTPA-octreotide and diminished upon receptor blocking. In all tissues except the kidneys, the uptake of 99mTc-RC-160 was also higher than that of 111In-DTPA-octreotide. The uptake of 99mTc-RC-160 was influenced by the amount of peptide injected and the best tumor/muscle and tumor/blood ratios were obtained when only one micrograms of the peptide (200 Ci/mmol) was administered.

Radiolabelled peptides : now and the future

Designed by nature for stimulating, inhibiting or regulating numerous life functions, for a long time peptides have been considered ideal agents for therapeutic applications. In recent years, radiolabelled peptides have emerged as a new, useful class of radiopharmaceuticals for diagnosis of a variety of endocrine tumours. Although clinical results with 111In- and 123I-labelled peptides have been excellent, extensive efforts have been made in preparing and evaluating peptides labelled with 99Tcm. This is the radionuclide of choice by virtue of its cost-effectiveness, availability and desirable nuclear characteristics. With 99Tcm, certain peptides can be labelled ‘directly’ without a loss of functional specificity, but certain peptides must be labelled using bifunctional chelating agents. The bifunctional chelating agent methods are chemically complex and frequently inefficient. Instant kit preparations are, however, possible.Important tests in the evaluation of these agents are receptor binding assays as well as examinations in experimental pre-clinical models. Peptides are small and can be taken up in target tissues non-specifically by increased capillary permeability. However, something that is frequently forgotten during such evaluations are control studies with non-specific peptides of similar size and charge. Radiolabelled peptides aimed at instant in vivo interaction with neutrophils or platelets must also be examined carefully to ensure that they do not induce neutropaenia or thrombocytopaenia. The preparation of radiolabelled peptides is not only challenging but also exciting, since these agents will continue to be promising and rewarding radiopharmaceuticals for decades to come.

Imaging experimental infective endocarditis with indium-111-labeled blood cellular components.

The capability of radionuclide imaging to detect experimental aortic valve infective endocarditis was assessed with indium-111 (111In)-labeled blood cells. Sequential cardiac imaging and tissue distribution studies were obtained in 17 rabbits with infective endocarditis after administration of 111In-platelets and in five after 111In-polymorphonuclear leukocytes. Forty-eight to 72 hours after platelet administration, in vivo imaging demonstrated abnormal 111In uptake in all animals in the region of the aortic valve in an anatomically distinct pattern. Images of the excised heart showed discrete cardiac uptake conforming to the in vivo image and gross pathological examination. 111In platelet uptake in vegetations from the 17 animals averaged 240 ± 41 times greater than that in normal niyocardium and 99 ± 15 times greater uptake in blood. In contrast, 111In-leukocyte cardiac imaging showed no abnormal aortic valve uptake 24 hours after tracer administration and the lesion myocardium activity ratio was only 5 ± 2 (3 ± 1 for lesion/blood activity). Four normal rabbits demonstrated neither positive 111.In platelet scintigraphs nor abnormal cardiac tissue uptake. Likewise, noncellular 111In was not concentrated to any significant extent in three animals with infective endocarditis. This study demonstrates that 111In platelet, but not leukocyte cardiac imaging, is a sensitive technique for detecting experimental infective endocarditis. The imaging data conform to the cellular pathology of the infective endocarditis vegetatio