Antiproliferative efficacy of the somatostatin analogue TT-232 in human melanoma cells and tumours.

BACKGROUND: TT-232, a somatostatin analogue, induces apoptosis in various tumours. The aim of our study was to characterise its effect on human melanoma cells and tumours. MATERIALS AND METHODS: Proliferation of seven melanoma cell lines was tested in vitro with the methylene blue test. D10 and 205 cells were also implanted into CB17-scid mice which received 30-150-750 micrograms/kg/day of TT-232 or saline. Animals with 205 cells received twice-daily subcutaneous injections whereas animals with D10 cells were treated with osmotic mini-pumps. In addition, TT-232 metabolites were generated with tissue homogenates and tested in vitro. RESULTS: TT-232 strongly inhibited proliferation of all cell lines in vitro and tumour growth in vivo. Two out of 8 animals (30-150 micrograms/kg) in the 205 model and one out of 8(150 micrograms/kg) in the D10 model became completely tumour-free at the 11th and 9th day of treatment, respectively. TT-232 was degraded only by liver homogenate whilst its metabolite had no antiproliferative effect in vitro. CONCLUSIONS: TT-232 is a promising drug candidate for melanoma.

Preclinical comparison in AR4-2J tumor-bearing mice of four radiolabeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-somatostatin analogs for tumor diagnosis and internal radiotherapy.

Somatostatin analogs labeled with radionuclides are of considerable interest in nuclear oncology as diagnostic or therapeutic tools for somatostatin receptor (SSTR)-expressing tumors. We investigated the suitability of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as a replacement for the widely used diethylenetriaminepentaacetic acid, to enable stable labeling of somatostatin analogs with both therapeutic (90Y) and diagnostic (111In) radionuclides. The three clinically relevant somatostatin agonists, octreotide, vapreotide, and lanreotide, together with the newly designed Tyr3-octreotide (TyrOc), were conjugated to DOTA and labeled with 90Y or 111In. For all DOTA-somatostatin analogs tested, irrespective of the incorporated radionuclide, we observed favorable biodistribution profiles in AR4-2J tumor-bearing mice: 1) a rapid clearance from all SSTR-negative tissues except kidney; 2) a specific uptake in SSTR-positive tissues, including tumor; and 3) an excellent tumor penetration. The main route of excretion was via the kidneys. Nevertheless, DOTATOC was clearly superior to the other DOTA-somatostatin analogs tested, as well as OctreoScan, as indicated by the highest tumor-to-nontarget-tissue ratio, including the tumor-to-SSTR-positive-tissue ratios. The presence of different SSTR subtypes in the SSTR-positive tissues possibly contributes to these differential uptakes. We assume that the very favorable behavior of DOTATOC in our mouse model makes this radioligand very promising for future applications in nuclear oncology.

Receptor targeting for tumor localisation and therapy with radiopeptides.

Receptor targeting with radiolabeled peptides has become very important in nuclear oncology in the past few years. The most frequently used peptides in the clinic are analogs of somatostatin (SRIF), e.g. OctreoScan, which contain chelators for the radioisotopes 111In, 86Y, 90Y, 67Ga, 68Ga and 64Cu or for 99mTc and 188Re. and were labelled with the halogens 123I and 18F. Radiolabeled analogs of &alpha-melanocyte-stimulating hormone (&alpha-MSH), neurotensin, vasoactive intestinal peptide (VIP), bombesin (BN), substance P (SP) and gastrin/cholecystokinin (CCK) are also being developed, evaluated in vitro and in vivo and tested for clinical application. This review focuses on the expression in tumors and the regulation of receptors for these neuropeptides as well as the development of novel chelator-peptide conjugates suitable for in vivo scintigraphy or internal radiotherapy. The state of the art of radiopeptide pharmaceuticals is illustrated with four SRIF analogs, modified with the macrocyclic chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA): [D-Phe1]-octreotide (DOTAOC), [D-Phe1, Tyr3]-octreotide (DOTATOC), vapreotide (DOTAVAP) and lanreotide (DOTALAN). DOTA is almost a universal chelator capable of strongly encapsulating hard metals such as 111In and 67Ga for Single Photon Emission Tomography (SPET), 68Ga, 86Y and 64Cu for Positron Emission Tomography (PET) as well as 90Y for receptor-mediated radionuclide therapy and radiolanthanides which exhibit different interesting decay schemes. From biodistribution studies in experimental animals and from clinical data it is concluded that DOTATOC is currently the most suitable SRIF radiopeptide with the best potential in the clinic.

Differential regulation of somatostatin receptor type 2 (sst 2) expression in AR4-2J tumor cells implanted into mice during octreotide treatment.

Octreotide is a somatostatin analogue that is widely used for cancer therapy and tumor imaging. Its efficacy in tumors depends mainly on the expression of the somatostatin receptor type 2 (sst 2). Desensitization and down-regulation of sst 2 after agonist exposure can have important consequences for patients under ongoing octreotide therapy because it may induce temporary tumor unresponsiveness and impair sst 2-based tumor scintigraphy. Therefore, we have investigated the effect of octreotide on sst 2 expression in vitro, as well as in a tumor mouse model. In vitro, short exposure to octreotide induced rapid dose-dependent down-regulation of sst 2 in the rat pancreatic AR4-2J cell line. Within 0.5 h, 80% of sst 2 had disappeared from the cell surface. A total recovery required 24 h and was shown to depend on protein synthesis, but not on new sst 2 mRNA transcription, indicating that sst 2 was probably degraded during the down-regulation process. Similar results were obtained in vivo. On the other hand, long-term continuous release of octreotide for 7 days, as achieved with octreotide-containing osmotic minipumps, caused sst 2 up-regulation in vivo, but not in vitro. Furthermore, this up-regulation of sst 2 in tumor-bearing scid mice was shown to depend on constant exposure of the animals to octreotide, as it was not observed when octreotide was given discontinuously in two s.c. daily injections. These results demonstrate that the continuous release of a small amount of octreotide, which in cancer therapy may be achieved with long-acting release formulations of the peptide, can induce sst 2 up-regulation on cancer cells. This may improve the efficacy of both tumor imaging and long-term octreotide therapy.

A microplate binding assay for the somatostatin type-2 receptor (SSTR2).

The clinical importance of somatostatin type-2 receptors (SSTR2) and the study of novel analogues of somatostatin such as OctreoScan or [Tyr3]-octreotide containing DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal chelator led us to develop a methodology to monitor the expression of SSTR2 on tumours of pancreatic origin (e.g. rat AR4-2J cancer cells). Usual binding assay protocols using the commercial [125I][Tyr1]-somatostatin radioligand failed, even in the presence of a cocktail of protease inhibitors with a broad spectrum of activity, possibly due to the high susceptibility of this tracer to proteases expressed in pancreatic cells. We prepared our own radioligand [125I][Tyr2]-octreotide which was shown to be much more resistant to degradation after incubation with AR4-2J plasma membranes. As expected, the increased stability of [125I][Tyr3]-octreotide was associated with good binding to SSTR2. Addition of appropriate protease inhibitors further increased the specific binding of [125I][Tyr3]-octreotide to AR4-2J plasma membranes without affecting the stability of the tracer, suggesting that the protease inhibitors also protect the integrity of SSTR2. Optimal conditions (time, temperature, medium) were developed for a binding assay in 96-well plates using AR4-2J plasma membranes in order to make the assay suitable for high-throughput analysis. This protocol was the basis for studying the in vivo regulation of SSTR2 expression in AR4-2J cells implanted into scid mice after exposure to different compounds.

A combinatorial peptoid library for the identification of novel MSH and GRP/bombesin receptor ligands.

A tripeptoid library was synthesized using 69 different primary amines in initially 69 individual reactions by the mix and split approach. The resulting library consisted of 328,509 (69(3)) single compounds, divided in 69 subpools each containing 4,761 entities. The 69 subpools were tested in two binding assays, one for alpha-MSH (alpha-melanotropin) and one for GRP (gastrin-releasing peptide)/bombesin. The sublibraries with the highest affinity to the MSH receptor (i.e. melanocortin type 1 or MC1 receptor) and, respectively, the GRP-preferring bombesin receptor were identified by an iterative process. Individual tripeptoids with good binding activity were resynthesized, analyzed and their dissociation constants and biological activity determined. The KD of the most potent MC1 receptor ligand was 1.58 mumol/l and that of the GRP-preferring bombesin receptor 3.40 mumol/l. Extension of this latter tripeptoid structure whose KD value increased to 280 nmol/l. A similar increase in activity was not observed with the most potent MSH tripeptoid ligand when extended by one residue, but a compound suitable for radioiodination and lacking the N-terminal amino group had a slightly higher binding activity than the tripeptoids (KD approximately 850 nmol/l). These results demonstrate that testing a peptoid library containing 328,509 single compounds led to the successful identification of new ligands for both the MC1 receptor as well as the GRP-preferring bombesin receptor.

Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4.

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.

Lymphotoxicity and myelotoxicity of doxorubicin and SDZ PSC 833 combined chemotherapies for normal mice.

In the mouse, the P-glycoprotein-directed chemosensitizer SDZ PSC 833 could both restore a therapeutic window for doxorubicin against multidrug-resistant tumors, by inhibiting P-glycoprotein function, and increase the anti-cancer drug efficacy against drug-sensitive tumors, by increasing doxorubicin bioavailability. Since the success of such combined chemotherapy treatments might have been limited by the myelotoxicity of doxorubicin and the P-glycoprotein expression on some blood cells, their lymphotoxicity and myelotoxicity was studied on normal B6D2F1 mice, and whenever possible, the persistence of blood cell alterations was also searched for in scid recipients of lymphohaematopoietic grafts from the donor mice. Analyzed parameters were blood, lymphoid and myeloid cell numbers, proliferative responses to T- and B-cell mitogens, and serum immunoglobulin levels. Cell alterations caused by doxorubicin alone were potentiated by SDZ PSC 833, but did not persist in scid recipients. Chemotherapy regimens combining SDZ PSC 833 and doxorubicin, and known for their therapeutic benefit for multidrug-resistant tumor-bearing mice, only caused a rather mild toxicity for the lympho-myeloid system of normal mice.

Myeloid and lymphoid cell alterations in normal mice exposed to chemotherapy with doxorubicin and/or the multidrug-resistance reversing agent SDZ PSC 833.

The cyclosporin SDZ PSC 833 (PSC) is a potent in vivo chemosensitizer for tumor cells with P-glycoprotein(Pgp)-dependent multidrug resistance (MDR). However, Pgp expression also occurs in CD8+ T cells, NK cells, macrophages and stem cells. In order to find whether PSC might display specific myelotoxicity or potentiate the toxicity of anti-cancer drugs, healthy mice were exposed to single doxorubicin (DOX) and combined (DOX + PSC) chemotherapy protocols known to be near or above the borderline of toxicity for tumor-bearing mice. Mice treated with DOX alone or with (DOX + PSC) showed transient spleen hypoplasia, with a general decrease of all leucocyte lineages and a persistent fall in the numbers of B cells in the bone marrow. In (DOX + PSC)-treated mice, PSC only potentiated the DOX effects without inducing specific depletions of the Pgp-expressing leukocytes (CD8+ and Mac-I+ cells). Hematopoietic cell grafts from normal mice to (DOX +/- PSC)-treated mice did not correct their B-cell lineage deficiency. When lethally irradiated mice were rehabilitated with hematopoietic cells from (DOX +/- PSC)-treated mice (including those with very reduced survival), all chimeras survived for at least 8 months after the cell graft, at which time their leucocyte population profiles were similar to those of control chimeras.

gld and lpr hematopoietic cell transfers: common and different serological features of the C57BL/6 chimeras.

Homozygosity for either the lpr (lymphoproliferation) or the gld (generalized lymphoproliferative disease) mutation in mice causes the development of strikingly similar hyperglobulinemia and lymphoproliferative syndromes. Nevertheless, previous studies of various C57BL/6 chimeras obtained by reconstitution of irradiated recipients with hematopoietic cells (HC), differing at the bg, gld, lpr, and/or nu loci, showed that the lpr and gld syndromes had distinct etiologies. The [lpr-->lpr], [gld-->gld], and [gld-->wild] chimeras developed lymphoid hyperplasia, while the [lpr-->wild, bg, or gld] and [nulpr-->wild or bg] chimeras developed a severe persistent lymphoid aplasia. We now show that the serological status (immunoglobulin (Ig) levels and Ig isotype distribution) of the [lpr-->lpr], [gld-->gld], and [gld-->wild] chimeras were roughly equivalent to those of genetic lpr and gld mice. Despite their lymphoid aplasia, all the [lpr-->non-lpr] chimeras displayed surprisingly normal serum Ig levels, similar to [wild-->wild] control chimeras, although always with some abnormal isotype profile. In fact, an early but transient increase of serum IgG1 levels was found in all [lpr-->wild, bg, or gld], [lpr-->lpr], [nulpr-->wild or bg], [wild-->lpr], and [gld-->wild or gld] types of chimeras. Despite a common early behavior, the host type and/or the gld or lpr HC origin may cause later divergences of the gld or lpr HC grafted chimeras.

Adoptive transfer of the entire gld (generalized lymphoproliferative disease) syndrome in nude beige mice by a single gld thymocyte graft.

C57BL/6 nude beige mice (B6 nubq; no T cell, no NK activity) were used as recipients for the adoptive transfer of thymocytes from B6 gld mice (generalized lymphoproliferative disease) which are a model of systemic lupus erythematous. The [gld----nubg] chimeras showed several similarities with gld control mice including the T cell disorders (lymphoproliferation and Con A-response deficiency of splenocytes) and B cell disorders (hyperglobulinemia and elevated anti-single-stranded DNA antibody titers). This suggests that the gld lymphoproliferative disorder has a thymic origin (and does not result from an abnormally extrathymic T cell development) and that the gld T cells have an essential role for the emergence of the disorders of both the T and B cells.

Adoptive transfer of the generalized lymphoproliferative disease (gld) syndrome in nude beige mice.

C57BL/6 nude beige mice (B6 nubg) were used as recipients for the transfer of haematopoietic cells from either B6 wild as control mice, or systemic lupus erythematous B6 mice homozygous for the recessive generalized lymphadenopathy disease (gld) locus. Both gld and wild cell grafts prolonged survival of the short-living B6 nubg recipients and restored some T-cell functions, as monitored by the presence of T-dependent Ig isotypes in the serum and responsiveness of spleen cells to a T-cell mitogen. Moreover, the [gld----nubg] chimeras but not the [wild----nubg] chimeras showed several similarities with gld control mice, particularly, a spleen and lymph node hyperplasia, elevated anti-single-stranded DNA antibody titres and a hyperglobulinaemia. This hyperglobulinaemia was however qualitatively different from the gld-type hyperglobulinaemia with an important contribution of the IgG1 isotype; the lymph node hyperplasia was also less marked than in B6 gld mice.

Adoptive transfer of the gld syndrome in double congenic nude lpr mice.

Homozygosity for either the lymphoproliferation (lpr) or the generalized lymphoproliferative disease (gld) mutation of mice causes the development of strikingly similar autoimmune and lymphoproliferative syndromes. The relationship between the lpr and gld mutations was studied by grafting B6 gld spleen cells (SC) to athymic B6 nude lpr mice (B6 nulpr) or to B6 nude (B6 nu) mice as controls. The injection of B6 gld SC, but not of B6 wild SC, to B6 nulpr mice caused a prolongation of survival of the short living B6 nulpr recipients. This was associated with elevated anti-single stranded DNA antibody titers and a serum hyperglobulinemia, as well as by a splenomegaly which was nearly as high as in genetically B6 gld mice, and by a marked lymphadenopathy (though milder than that of B6 gld mice). In contrast the [gld----nu] chimaeras showed a more attenuated form of gld-induced syndrome. These results suggest that the lpr environment supplied in athymic lpr recipients is compatible with--and may even favour--the development of the gld-induced syndrome.

A quick procedure for identifying doubly homozygous immunodeficient scid beige mice.

This article describes a rapid and reliable procedure for identifying mice which are doubly homozygous at the scid and beige (bg) loci starting from CB17 scid (no T and B cells) and B6 bg mice (no NK activity). The [scid, bg] mice are directly identified in the F2 progeny by monitoring (1) the hypogammaglobulinemia for the scid gene and (2) the prolonged bleeding associated with the bg gene. Like CB17 scid mice, the [scid, bg] mice show a high susceptibility to infections and die early in life unless they are protected against potential infections. This is achieved by a graft of splenocytes plus bone marrow cells from (B6 bg x CB17 scid) F1 mice. These [scid, bg] mice combine the bg and scid immunodeficiencies and should be better recipients for xenografts than classical scid mice.

Different nature of the proliferation defects of GLD, LPR and MEV C57BL/6 mouse lymphoid cells.

The three non-allelic gld, lpr and mev mutations in the mouse all lead to profound immunodeficiency besides a splenomegaly and a generalized autoimmunity. Spleen cells from young B6 gld, B6 lpr and B6 mev mice all display a decreased proliferative response to the T-cell mitogen concanavalin A (ConA), but the nature of the deficiency seems very different. No restoration of proliferation could be obtained by adding exogenous recombinant rIL2 to ConA-treated mev spleen cells, this lack of IL2-responsiveness suggesting a lack of (functional) IL2-receptors. In young mice of both gld and lpr strains, a B6 wild-type level of proliferation could be reached by rIl2 addition to ConA-treated spleen cells, this normal responsiveness to exogenous IL2 suggesting a normal expression of IL2-receptors. The endogenous IL2 production by ConA-treated spleen cells decreased very much with ageing in both B6 gld and B6 lpr mice. Yet, IL2 production in young mice revealed an earlier deficiency of the B6 lpr mice: the young B6 gld IL2 levels reached about 60% of age-matched B6 wild cell levels, but the B6 lpr levels reached 14% only. Finally the addition of exogenous rIL2 to ConA-pretreated cells from old B6 gld and B6 lpr mice, while enhancing the proliferative responses, could not restore the B6 wild-type levels. This suggests that, with ageing, the expression of functional IL2-receptors may become as abnormal in these gld and lpr mutants as it is from birth in the mev mutant mice.