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Original Article |
Interactions Cellulaires Neuroendocriniennes, Unité Mixte de Recherche 6544, Centre National de la Recherche Scientifique (A.S., E.L., G.G., A.B., A.E., P.J.) Institut Fédératif Jean Roche, Faculté de Médecine Nord, 13916 Marseille Cedex 20, France; and Biomeasure Inc. (S.K., J.E.T., M.D.C.), Milford, Massachusetts 01757
Address all correspondence and requests for reprints to: Philippe Jaquet, Interactions Cellulaires Neuroendocriniennes, Unité Mixte de Recherche 6544, Centre National de la Recherche Scientifique, Institut Fédératif Jean Roche, Faculté de Médecine Nord, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France. E-mail: jaquet.p{at}jean-roche.univ-mrs.fr.
Abstract
In acromegaly, the combination of somatostatin (SS) and dopamine (DA) agonists has been shown to enhance suppression of GH secretion. In the present study, a new chimeric molecule, BIM-23A387, which selectively binds to the SS subtype 2 receptor (sst2; Ki = 0.10 nM) and to the DA D2 receptor (D2DR; Ki = 22.1 nM) was tested in cultures prepared from 11 human GH-secreting tumors for its ability to suppress GH and prolactin (PRL) secretion. The chimeric compound was compared with individual sst2 and D2DR agonists of comparable activity at the individual receptors. All tumors expressed both sst2 and D2DR mRNAs (0.8 ± 0.2 and 4.7 ± 0.7 copy/copy ß-glucuronidase mRNA, respectively). In cell cultures from seven octreotide-sensitive tumors, the maximal inhibition of GH release induced by the individual sst2 and D2DR analogs and by BIM-23A387 was similar. However, the mean EC50 for GH suppression by BIM-23A387 (0.2 pM) was 50 times lower than that of the individual sst2 and D2DR analogs, either used individually or combined. Similar data were obtained in four tumors that were only partially responsive to octreotide. The inhibition of GH release by BIM-23A387 was only partially reversed by the D2R2 antagonist, sulpiride, or by the sst2 antagonist, BIM-23454. Only when both antagonists were combined was the GH suppressive effect of BIM-23A387 totally reversed. Finally, BIM-23A387 produced a mean 73 ± 6% inhibition of PRL in six mixed GH plus PRL tumors. These data demonstrate an enhanced potency of the chimeric molecule, BIM-23A387, in suppressing GH and PRL secretion from acromegalic tumors, which cannot be explained merely on the basis of binding affinity for SS and/or DA receptors.
OVER THE YEARS, the criteria by which acromegaly is considered fully controlled have become more stringent. From recent consensus studies, a mean GH plasma value less than 2.5 µg/liter, associated with IGF-I levels within the normal range for sex and age, is required to demonstrate full efficacy of treatment (1). This goal is achieved in 45% of patients treated with three sc injections of the somatostatin (SS) analog, octreotide (2, 3, 4). Treatment with the sustained-release, depot preparations of Sandostatin LAR or of SR-Lanreotide, which are biodegradable polymer microsphere formulations of octreotide and lanreotide, respectively, achieves adequate control of GH and IGF-I levels in about 60% of acromegalic patients (5, 6). Even with the sustained-release preparations, the efficacy remains only partial in at least one third of patients. Because dopamine (DA) agonists have also been shown to inhibit GH hypersecretion in 20% of acromegalic patients (7), the rationale for their combination with SS analogs has been proposed for some time (8). Until now, the efficacy of such combined treatment has not been firmly established due to controversial data obtained in a limited number of patients (9). Very recently, it was reported that cotransfection of the SS subtype 5 receptor (sst5) and the DA D2 receptor (D2DR) led to a heterodimeric association between the receptors with enhanced biological activity (10). If the formation of such a heterodimer can be regulated by specific ligands, it could be of potential interest for the management of GH-secreting pituitary tumors that coexpress SS and DA receptors. Recently, a chimeric molecule, BIM-23A387, was created that contains structural elements of both SS and DA. The hybrid molecule retains potent, selective agonist activity at both the SS subtype 2 receptor (sst2) and the D2DR. The main objectives of this study were to compare the actions of the sst2 and D2DR agonists, to examine possible additive or synergistic effects of combined sst2 and D2DR activation, and to compare and evaluate the chimeric SS-DA molecule, on GH and prolactin (PRL) release from tumors expressing varying degrees of both sst2 and D2DR receptor mRNAs.
Subjects and Methods
The present study was approved by the ethics committee of the University of Aix-Marseilles (Marseilles, France). Eleven acromegalic patients (six women and five men), aged 37 ± 4 yr, presenting with macroadenoma were studied. Their endocrine status and the neuroradiological characterization of the pituitary adenomas were documented before treatment. Basal GH levels were the mean of eight consecutive measurements obtained hourly between 0800 and 1300 h. SS agonist and DA agonist sensitivity were assessed on different days by an acute test using octreotide and quinagolide, respectively. The octreotide test (Sandostatin, Novartis Pharmaceuticals, Basel, Switzerland) used a single sc 200-µg injection. Plasma GH levels were measured both before and 2–4 h after octreotide. The quinagolide test (Norprolac, Novartis Pharmaceuticals) used a single oral 150-µg administration, and plasma GH levels were measured both before and 2–4 h after quinagolide. According to the test results, seven patients were considered octreotide responders (mean GH suppression, 81 ± 4%), whereas the last four cases were considered partial octreotide responders (mean GH suppression, 39 ± 3%). In this series, most of the patients (8 of 11) presented with mild hyperprolactinemia (mean PRL basal value, 56 ± 12 µg/liter). Quinagolide testing was performed in 9 of 11 patients, with resulting mean GH suppression of 35 ± 9%. The lowest quinagolide inhibitory effects were observed in the same patients (A8–A11) previously classified as partially responsive to octreotide by in vivo testing. All patients underwent transsphenoidal surgery. After surgery, a portion of each tumor tissue was analyzed in terms of the quantitative expression of mRNA for the D2DR and for the sst2 and sst5 receptor subtypes. The remainder of the tissue was dispersed for cell culture to test the pharmacological effects of different analogs. The clinical endocrine and tumoral status of each patient and the GH and PRL responses to acute octreotide or quinagolide tests are summarized in Tables 1
and 2.
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GH and PRL were measured using commercial immunoradiometric kits (Immunotech, Marseilles, France). Normal GH values ranged from 0.2–2.4 µg/liter, and normal PRL values ranged from 1–24 µg/liter in women and 1–17 µg/liter in men. After an ethanol-acid extraction, plasma IGF-I was measured using the IGF-I RIA kit from Nichols Institute Diagnostics (San Juan Capistrano, CA). The normal ranges, according to sex and age, were established by our laboratory.
Detection of sst and D2DR mRNAs
Total RNA was extracted from 30–60 mg of tissue from each tumor using the RNA easy isolation system (QIAGEN, Courtaboeuf, France). One microgram of total RNA prepared from tumoral pituitary tissues was used for cDNA synthesis with 200 U Superscript II reverse transcriptase (Life Technologies, Inc. Cergy-Pontoise, France) primed with 300 ng random primer in 20 µl 50 mmol/liter Tris-HCl (pH 8.3), 75 mmol/liter KCl, 3 mmol/liter MgCl2, 0.5 mmol/liter of each dNTP, and 40 U RNasin (Promega Corp.). The reaction mix was incubated for 10 min at 25 C, followed by 50 min at 42 C.
The 5' exonuclease (Taq Man) assay, which produces a direct proportional readout for the progression of PCR, was used (11). Amplification of cDNA derived from 50–150 ng total RNA was performed in a 25-µl reaction volume with 300 nM of each primer, 200 nM of the probe, and 12.5 µl MasterMix (PE Applied Biosystems, Paris, France). The probe comprised 20–30 nucleotides with 5' end substitution with a fluorophore and a quencher substitution at the 3' end. The synthetic sst and D2DR cDNA primers used in the PCR were 19- or 20-mers as follows: sst2 (GenBank accession no. M81830), sense (10–29), antisense (109–91), and probe (58–32); sst5 (GenBank accession no. L14865), sense (1103–1119), antisense (1156–1139), and probe (1137–1121); D2DR (GenBank accession no. AF050737 coding sequence), sense (1062–1083), antisense (1177–1156), and probe (1088–1115). The annealing-extension temperatures were 59 C for sst2, 63 C for sst5, and 60 C for D2DR. Forty cycles of two-step PCR-annealing extension, at specified temperatures, for 60 sec and denaturation at 95 C for 15 sec, were performed on an ABI Prism 7700 sequence detection apparatus (PE Applied Biosystems). The sst and D2DR mRNA levels were normalized to the ß-Gus mRNA levels obtained in the same reaction. The ß-Gus primers and probe were purchased from PE Applied Biosystems (chosen from the 11 housekeeping genes proposed, after preliminary experiments). For each measurement, three independent RT-PCR analyses were performed. To produce standard curves for sst mRNA, D2DR mRNA, and ß-Gus mRNA, cDNA constructs were produced for each parameter. Using the specific fluorogenic probes for each gene under the experimental conditions defined above, we obtained a linear relationship between the cDNA concentration and the threshold cycle of fluorescent signal for sst, D2DR, and ß-Gus from 50 to 5,000,000 copies cDNA target. For each unknown sample, we determined the threshold cycle values for all genes indicated, and the results were expressed as copies of sst or D2DR/copies of ß-Gus.
Cell culture studies
A portion of each tumor obtained at surgery was dissociated by enzymatic and mechanical methods. Depending on the tumor, 5–64 x 106 isolated cells were obtained. These cells were plated in multiwell culture dishes (Costar 3524, Costar, Brumath, France) that were coated with extracellular matrix from bovine endothelial corneal cells, as previously described (12), at a density of 2 x 104 cells per well. The DMEM supplemented with 2% fetal calf serum, antibiotics, insulin, transferrin, and selenium (12) was replaced every 3 d. After 6–8 d of culture, the effects of various doses of octreotide; quinagolide; the sst2-preferential agonist, BIM-23023; the D2DR agonist, BIM-53097, both alone and in combination; and the chimeric sst2/D2DR molecule, BIM-23A387, were tested for their ability to suppress GH and PRL release over an 8-h period. The experiments testing the ability of the sst2 antagonist, BIM-23454 (13), and the D2DR antagonist, sulpiride, to reverse GH suppression by the sst2 and D2DR analogs and the sst2 and D2DR chimeric compound, were performed on d 15 of culture. Such a delay was necessary to determine the dose-responses and the EC50 for GH inhibition by the different agonists. Because GH release is known to decrease as a function of time, the incubation period was extended to 12 h to ensure a significant GH concentration in the culture media. The details of these experiments are given in Results. Using these cell culture conditions, we have previously shown reproducible pharmacological responses to different stimuli for up to 30 d (12). We have also established a positive correlation between the level of sst2 mRNA expression and the degree of GH suppression induced by octreotide in vivo as well as in culture studies (11, 14 ). Each drug concentration was tested in quadruplicate.
Products
The BIM compounds were provided by Biomeasure, Inc. (Milford, MA). BIM-23023 is a highly preferential sst2 compound (sst2, Ki = 0.4 nM). Its EC50 for GH suppression (10 ± 8 pmol/liter) is 2-fold lower than that of octreotide as established in previous experiments (data not shown). BIM-53097 is a D2 DA agonist (D2DR, Ki = 28 nM). Its EC50 for GH suppression (100 ± 60 pmol/liter) is 10 times higher than that of quinagolide as established in previous experiments (data not shown). BIM-23A387 is a chimeric molecule that combines structural elements of both SS and DA and retains affinity for both the sst2 and D2DR receptors (sst2, Ki = 0.10 nM; D2DR, Ki = 22.1 nM). Octreotide and quinagolide were supplied by Novartis Pharmaceuticals. Sulpiride was purchased from Sigma Chemical Co. (Saint-Quentin Fallavier, France). The SS analogs were dissolved in 0.01 mol/liter acetic acid containing 0.1% purified human serum albumin (Life Technologies, Inc., Cergy-Pontoise, France). Quinagolide was initially prepared as a 10-3 mol/liter solution in 70% ethanol. All drugs were stored at -80 C as 10-3 mol/liter solutions. Fresh working solutions were prepared from a new aliquot for each experiment. The human sst and D2DR affinities (IC50; nanomoles per liter) were determined for the different compounds by radioligand receptor binding assays using membranes from transfected CHO-K1 cells expressing the human D2DR or the human sst subtypes as previously described (15). Results are summarized in Table 3.
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The results are presented as the mean ± SEM. Statistical significance between two unpaired groups was determined by the Mann-Whitney U test. To measure the strength of association between the pairs of variables without specifying dependencies, Spearman order correlations were used. A P value less than 0.05 was considered significant for all tests.
Results
D2DR and sst subtype mRNA expression
Real-time PCR quantitative analysis was performed on tumor fragments of the 11 adenomas. Expression of D2DR, sst2, and sst5 mRNA was found in all tumors. The mean sst5, sst2, and D2DR mRNA levels were 10.9 ± 3.2, 0.8 ± 0.2, and 4.7 ± 0.7 copy/copy ß-Gus, respectively. The high expression of sst5 mRNA vs. sst2 mRNA has been previously reported in GH-secreting adenomas and was not correlated to the response to octreotide (11). The individual patterns of sst2 mRNA and D2DR mRNA expression are presented in Fig. 1. In this series, tumors A8-A11 expressed very low levels of sst2 mRNA and were also classified as partially responsive to octreotide. The level of sst2 mRNA correlated to the percentage GH suppression in response to octreotide (P < 0.03). The D2DR mRNA was highly expressed in tumors A1, A2, A3, A5, A6, A7, and A11, which presented as mixed GH-PRL adenomas (mean value, 6.5 ± 0.9 copy/copy ß-Gus vs. 1.5 ± 0.4 copy/copy ß-Gus in the four pure GH-secreting tumors A4, A8, A9, and A10; P < 0.008). Tumors A8-A10, which expressed the lowest levels of D2DR mRNA, were the least responsive to quinagolide in terms of GH suppression. In the 11 tumors, the levels of D2DR mRNA and sst2 mRNA varied independently with each tumor.
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In studies of cultured cells derived from the 11 tumors, the effects of octreotide and quinagolide on GH secretion were measured after an 8-h incubation period. As shown in Fig. 2, the maximal inhibition of GH release induced by 10 nmol/liter octreotide, as compared with controls (medium alone), ranged from 57–10% among the 11 adenoma cell cultures. The mean GH suppression was 43 ± 4% in the cell cultures derived from tumors A1-A7 from patients considered responsive to octreotide. Using tumor cells from patients considered only partially responsive to octreotide (A8–A11), mean GH suppression by octreotide was 23 ± 7%. In the same experiments, the mean maximal inhibition of GH release by quinagolide, at 10 nmol/liter concentrations, was 41 ± 4% with the seven octreotide-responsive adenomas and 19 ± 6% with tumors from the four patients that were partially responsive to octreotide. Thus, most of the tumors showed similar maximal suppression of GH in response to treatment with either the SS or DA analog, which was correlated with the sst2 mRNA, but not with the D2DR mRNA levels.
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To know whether the combination of the DA agonist, BIM-53097, and the sst2- preferential agonist, BIM-23023, could produce a synergistic effect on GH inhibition, both drugs were tested at various doses, either alone or in combination, in the seven octreotide-responsive and the four octreotide-partially responsive adenoma cell cultures. As shown in Fig. 3, the mean maximal GH suppression achieved with BIM-53097 and BIM-23023, either alone or in combination, was similar among both the octreotide responsive tumors (43 ± 6%, 47 ± 6%, and 48 ± 4%) and the tumors partially responsive to octreotide (15 ± 5%, 21 ± 6%, and 22 ± 6%). In the octreotide-sensitive tumors, the sst2 compound BIM-23023 proved to be more potent than the D2DR compound, BIM-53097 (EC50 = 10 pmol/liter vs. 100 pmol/liter, respectively). When the D2DR and sst2 compounds were added together, the dose-related inhibition of GH release was similar to that produced by the most potent of the individual compounds. These data demonstrate the absence of additivity in the combination of sst2 and D2DR analogs on GH suppression.
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In the seven octreotide-responsive tumors, the GH suppressive effects of the sst2/D2DR chimeric molecule, BIM-23A387, were compared with the combination of the sst2- and D2DR-preferential compounds. As shown in Fig. 4, the mean dose-related pattern of GH suppression produced by BIM-23A387 (EC50 = 0.2 pmol/liter; range, 0.01–5.0 pmol/liter) was markedly distinct from that induced by the combination of BIM-23023 and BIM-53097 (EC50 = 8 pmol/liter; range, 2–50 pmol/liter), although, at nanomolar concentrations, the mean maximal GH suppression was similar (52 ± 4% with BIM-23A87 and 48 ± 4% with the combination of BIM-23023 and BIM-53097). One tumor (A4) differed from the others because it had high levels of sst2 mRNA expression with low D2DR mRNA expression (Fig. 1
) and low maximal GH suppression by quinagolide (Fig. 2). In this case, the chimeric molecule, BIM-23A387, produced a dose-related suppression of GH release that was similar to that achieved by the sst2 compound, BIM-23023. In the cell cultures from adenomas partially responsive to octreotide, the same distinct, dose-related pattern of GH suppression was also observed [BIM-23A387, EC50 = 2 pmol/liter (range, 1–10), vs. BIM-23023 + BIM-53097, 50 pmol/liter (range, 15–200)]. In these partially responsive tumors, as with the octreotide-sensitive tumors, the BIM-23A387 chimera and the combined individual sst2 and D2DR-preferential compounds both produced similar maximal suppression of GH when tested in the nanomolar concentration range.
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Using cells cultured from tumor A7, the effects of the D2DR antagonist, sulpiride (10 µmol/liter), and of the sst2 antagonist, BIM-23454 (100 nmol/liter; Ref.13), were tested. At these concentrations, neither of the antagonists had any effect upon basal GH or PRL secretion (data not shown). In these experiments, the sst2- preferential compound, BIM-23023; the D2DR agonist, BIM-53097; and the sst2-D2DR chimera, BIM-23A387, were used at concentrations previously determined to correspond to their EC50 for GH or PRL suppression. The cells were incubated for a 12-h period in the presence of each of the agonists, either alone or with sulpiride, BIM-23454, or both antagonists in combination. As shown in Fig. 5, GH suppression with a 10 pmol/liter concentration of the sst2-preferential compound, BIM-23023, was totally reversed by the sst2 antagonist, BIM-23454. The D2DR antagonist was ineffective in reversing the action of BIM-23023. Similarly, the GH suppression obtained with a nanomolar concentration of the D2DR agonist, BIM-53097, was totally reversed by sulpiride, whereas the sst2 antagonist, BIM-23454, had no effect. In the presence of the BIM-23A387 chimera, at a 1 pmol/liter concentration, GH release was suppressed by 31%. This effect was only partially reversed by the sst2 and D2DR antagonists, BIM-23454 and sulpiride, added individually. Only when both antagonists were combined was the GH suppression produced by the BIM-23A387 chimera completely reversed. Similar results were also obtained by examining PRL release in the same experiments (data not shown). These data demonstrate that the chimeric molecule, BIM-23A387, can suppress hormone release through both the sst2 and D2DR receptor pathways.
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The dose-related inhibition of PRL secretion by BIM-23023 and BIM-53097, either alone or in combination, and by BIM-23A387 was analyzed in tumor cell cultures from six different tumors (A1–A3 and A5–A7). As shown in Fig. 6A, the dose-related inhibition of PRL release with increasing concentrations of BIM-23023 and BIM-53097 was similar, either alone or combined. Again, these data show the absence of additivity in the combination of sst2 and D2DR analogs on PRL suppression. In comparison, the sst2-D2DR chimera, BIM-23A387, was more effective in suppressing PRL secretion (mean EC50 = 0.9 pmol/liter and 40 pmol/liter with BIM-23A387 and BIM-23023 + BIM-53097, respectively; P < 0.04).
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Several groups have looked for a possible beneficial effect of the coadministration of SS and DA agonists on the inhibition of plasma GH levels in acromegalic patients. In a short-term study of 51 acromegalics (16), bromocriptine and octreotide coadministration enhanced GH suppression only 7–10 h after drug administration. From these data, no synergy was achieved by combining the two drugs. In several long-term studies of small cohorts of acromegalic patients, the combination of bromocriptine and octreotide was considered to be more effective (17, 18, 19, 20) in suppressing GH as compared with the results achieved by octreotide alone. However, at least one study reported no observable difference (21). More recently, the combination of more potent DA agonists, quinagolide or cabergoline, with depot preparations of SS agonists allowed normalization of IGF-I plasma levels in 6 of 17 cases (22, 23). Taken together, these data indicate that mainly in patients with mixed adenomas that cosecrete GH and PRL and in patients with only moderate elevation of plasma IGF-I levels, the combination of DA and SS drugs could be more effective than treatment with either drug alone (24).
Our in vitro studies have shown for the first time the lack of direct additivity at the cellular level between an sst2-preferring analog, BIM-23023, and a D2DR agonist, BIM-53097, upon suppression of GH release in cultured human adenoma cells. In all individual cell culture studies, the dose-response inhibition of GH and PRL secretion by the combination of SS and DA analogs was similar to that achieved by the most potent of the individual compounds, predominantly the sst2-preferential analog, BIM-23023. Among the four cell cultures (A8–A11) showing only a partial inhibitory effect of octreotide or BIM-23023 upon GH release, the real-time RT-PCR analysis evidenced in three of four cases a low expression of the D2DR receptor mRNA together with a low expression of sst2 mRNA (Fig. 1). Again, no significant additive effect of the combination of BIM-23023 and BIM-53097 was observed with the four cell cultures derived from tumors that were only partially responsive to octreotide or BIM-23023. In contrast, when the dopaminergic and somatostatinergic pharmacological activities were present within the same chimeric molecule, BIM-23A387, the results obtained were markedly different. In all but one of the tumors studied, based upon its EC50, BIM-23A387 was more potent than either BIM-23023 (sst2) or BIM-53097 (D2DR), either alone or in combination, in suppressing both GH and PRL secretion. This enhanced activity cannot be explained by a change in the affinity for either the sst2 or D2DR, because the individual affinity of the chimeric molecule for each of these receptors is roughly equivalent to those of BIM-23023 and BIM-53097, as shown in Table 3.
The reason for the enhanced potency of the chimeric molecule, BIM-23A387, is not yet known. One hypothesis may be oligomerization of the DA and SS receptors creating a novel receptor with distinct functionality. The ligand-induced oligomerization of G protein-coupled receptors has now been demonstrated for different receptors (25, 26). SS receptor subtypes have also been shown to homodimerize or heterodimerize (27, 28). Heterodimers resulting from interaction of the sst1 and sst5 subtypes showed increased ligand affinity and modified sst functionality (27). In contrast, when sst2 and sst3 were coexpressed in human embryonic kidney 293 cells, heterodimerization of the receptor subtypes resulted in inactivation of the sst3 subtype (28). The D2DR receptors have been shown to exist as homodimers in both rat and human brain tissues (29). These D2DRs can also form heterodimers with other G protein-coupled receptors such as the D3 receptor (30) or the sst5 subtype (10). In this latter example, cotransfection of D2DR and sst5 in CHO-K1 cells produced increased ligand binding affinity as well as new functional activities (10). In the example of DA-SS receptor heterodimers, the absence of preformed heterodimers (before ligand binding) implies the ligand-mediated regulation of these complexes. Other examples of ligand-induced G protein-coupled receptor heterodimers have been reported recently with the opioid receptors (31, 32) and with 
-aminobutyric acid (GABA) receptor subtypes (33, 34). In both examples, heterodimerization was responsible for an increase in biological functions mediated by the receptor. The example of GABA receptors is of particular interest because the two GABA receptor subtypes are inactive, and transduction of signals has been shown to exist only after coexpression and heterodimerization of GABABR1 and GABABR2 (34). In the case of opioid receptors, heterodimerization of the 
and 
receptors produces a modification in the ligand binding profiles as well as a synergistic inhibition of adenylyl cyclase activity (31). At present, the mechanism by which the DA-SS chimera produces GH suppression with a 50-fold lower EC50 than either DA agonists, SS agonists, or the combination of the two is unknown. However, the inability of either SS or DA antagonists to individually reverse the GH suppression induced by BIM-23A287, whereas both antagonists combined can achieve full reversal, suggests that the enhanced ability to modify the signaling properties of the receptors may be dependent on having the binding requirements for each receptor within the same molecule.
Finally, because the chimeric compound, BIM-23A387, is capable of producing potent suppression of both GH and PRL release, could it be a new candidate drug in the treatment of acromegaly? The high level of D2DR mRNA expression measured in 7 of 11 of our adenomas may introduce a bias in our analysis. The majority of the tumors, in the present study, presented as mixed tumors that were equally responsive to the inhibitory effects of DA and SS analogs. In an unselected population of acromegalic patients, the proportion of such mixed tumors is reported to represent only 36% of cases, according to the current functional classification of pituitary tumors (35). In pure GH-secreting adenomas, the density of DA receptors is low, and the inhibitory effects of DA agonists upon GH plasma levels are weak, if any (36). This was the case in 4 of 11 of the adenomas in the present study, which showed the lowest expression of both D2DR and sst2 mRNA and were among the tumors the least responsive to quinagolide as well as octreotide. However, although the results from this study suggest that the chimeric SS-DA compound will not display its enhanced potency with tumors that do not express D2DR, they also suggest that the chimera will be more efficacious than its SS component in tumors expressing adequate levels of sst2 and D2DR. Indeed, the data from our cell culture studies have to be extended by in vivo studies. Particularly, it is of interest to know whether the inclusion of the dopaminergic moiety could prolong the pharmacological properties of the chimeric molecule. Also, further studies of sst2 and D2DR expression and functionality in a larger population of acromegalic tumors are needed to better define the population of patients that could benefit from a SS-DA chimeric molecule as a therapeutic option for acromegaly.
Acknowledgments
We are grateful to Drs. H. Dufour and P. Paquis, neurosurgeons who provided the tumoral material. The skillful help of Mrs. C. Taverna in the redaction of the manuscript was greatly appreciated.
Footnotes
This work was supported by a grant from Biomeasure, Inc. (Milford, MA) and by Region Provence-Alpes-Côte d’Azur (Marseille, France).
Abbreviations: D2DR, DA D2 receptor; DA, dopamine; GABA, -aminobutyric acid; PRL, prolactin; SS, somatostatin; sst2, SS subtype 2 receptor.
Received June 14, 2002.
Accepted August 19, 2002.
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D. O'Toole, A. Saveanu, A. Couvelard, G. Gunz, A. Enjalbert, P. Jaquet, P. Ruszniewski, and A. Barlier The analysis of quantitative expression of somatostatin and dopamine receptors in gastro-entero-pancreatic tumours opens new therapeutic strategies Eur. J. Endocrinol., December 1, 2006; 155(6): 849 - 857. [Abstract] [Full Text] [PDF]
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 M. P. Gillam, M. E. Molitch, G. Lombardi, and A. Colao Advances in the Treatment of Prolactinomas Endocr. Rev., August 1, 2006; 27(5): 485 - 534. [Abstract] [Full Text] [PDF]
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 D. FERONE, M. BOSCHETTI, E. RESMINI, M. GIUSTI, V. ALBANESE, U. GOGLIA, M. ALBERTELLI, L. VERA, F. BIANCHI, and F. MINUTO Neuroendocrine-Immune Interactions: The Role of Cortistatin/Somatostatin System Ann. N.Y. Acad. Sci., June 1, 2006; 1069(1): 129 - 144. [Abstract] [Full Text] [PDF]
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 D. Ferone, M. Arvigo, C. Semino, P. Jaquet, A. Saveanu, J. E. Taylor, J.-P. Moreau, M. D. Culler, M. Albertelli, F. Minuto, et al. Somatostatin and dopamine receptor expression in lung carcinoma cells and effects of chimeric somatostatin-dopamine molecules on cell proliferation Am J Physiol Endocrinol Metab, December 1, 2005; 289(6): E1044 - E1050. [Abstract] [Full Text] [PDF]
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 M C Zatelli, D Piccin, F Tagliati, A Bottoni, M R Ambrosio, A Margutti, M Scanarini, M Bondanelli, M D Culler, and E C d. Uberti Dopamine receptor subtype 2 and somatostatin receptor subtype 5 expression influences somatostatin analogs effects on human somatotroph pituitary adenomas in vitro J. Mol. Endocrinol., October 1, 2005; 35(2): 333 - 341. [Abstract] [Full Text] [PDF]
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P Jaquet, G Gunz, A Saveanu, H Dufour, J Taylor, J Dong, S Kim, J-P Moreau, A Enjalbert, and M D Culler Efficacy of chimeric molecules directed towards multiple somatostatin and dopamine receptors on inhibition of GH and prolactin secretion from GH-secreting pituitary adenomas classified as partially responsive to somatostatin analog therapy Eur. J. Endocrinol., July 1, 2005; 153(1): 135 - 141. [Abstract] [Full Text] [PDF]
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 C. Roche, A. J Zamora, D. Taieb, E. Lavaque, R. Rasolonjanahary, H. Dufour, C. Bagnis, A. Enjalbert, and A. Barlier Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters J. Endocrinol., October 1, 2004; 183(1): 217 - 233. [Abstract] [Full Text] [PDF]
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S.-G. Ren, S. Kim, J. Taylor, J. Dong, J.-P. Moreau, M. D. Culler, and S. Melmed Suppression of Rat and Human Growth Hormone and Prolactin Secretion by a Novel Somatostatin/Dopaminergic Chimeric Ligand J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5414 - 5421. [Abstract] [Full Text] [PDF]
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M. C. Zatelli, D. Piccin, F. Tagliati, M. R. Ambrosio, A. Margutti, R. Padovani, M. Scanarini, M. D. Culler, and E. C. degli Uberti Somatostatin Receptor Subtype 1 Selective Activation in Human Growth Hormone (GH)- and Prolactin (PRL)-Secreting Pituitary Adenomas: Effects on Cell Viability, GH, and PRL Secretion J. Clin. Endocrinol. Metab., June 1, 2003; 88(6): 2797 - 2802. [Abstract] [Full Text] [PDF]
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L. J. Hofland and S. W. J. Lamberts The Pathophysiological Consequences of Somatostatin Receptor Internalization and Resistance Endocr. Rev., February 1, 2003; 24(1): 28 - 47. [Abstract] [Full Text] [PDF]
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