Maintained partial protection against Streptococcus pneumoniae despite B-cell depletion in mice vaccinated with a pneumococcal glycoconjugate vaccine.

OBJECTIVES: Anti-CD20 monoclonal antibody therapy rapidly depletes > 95% of CD20+ B cells from the circulation. B-cell depletion is an effective treatment for autoimmune disease and B-cell malignancies but also increases the risk of respiratory tract infections. This effect on adaptive immunity could be countered by vaccination. We have used mouse models to investigate the effects of B-cell depletion on pneumococcal vaccination, including protection against infection and timing of vaccination in relation to B-cell depletion. METHODS: C57BL/6 female mice were B-cell depleted using anti-CD20 antibody and immunized with two doses of Prevnar-13 vaccine either before or after anti-CD20 treatment. B-cell repertoire and Streptococcus pneumoniae-specific IgG levels were measured using whole-cell ELISA and flow cytometry antibody-binding assay. Protection induced by vaccination was assessed by challenging the mice using a S. pneumoniae pneumonia model. RESULTS: Antibody responses to S. pneumoniae were largely preserved in mice B-cell depleted after vaccination resulting in full protection against pneumococcal infections. In contrast, mice vaccinated with Prevnar-13 while B cells were depleted (with > 90% reduction in B-cell numbers) had decreased circulating anti-S. pneumoniae IgG and IgM levels (measured using ELISA and flow cytometry antibody binding assays). However, some antibody responses were maintained, and, although vaccine-induced protection against S. pneumoniae infection was impaired, septicaemia was still prevented in 50% of challenged mice. CONCLUSIONS: This study showed that although vaccine efficacy during periods of profound B-cell depletion was impaired some protective efficacy was preserved, suggesting that vaccination remains beneficial.

Effect of Subcutaneous Anti-CD20 Antibody-Mediated B Cell Depletion on Susceptibility to Pneumocystis Infection in Mice.

Prior work has shown that parenterally administered anti-CD20 (5D2) inhibits CD4+ T cell priming in response to challenge with Pneumocystis murina and predisposes to pneumonia. In this study, we investigated the effect of subcutaneous anti-CD20 antibody and Pneumocystis infection. In mice with primary infection, anti-CD20 antibody treatment depleted both CD19+ and CD27+ CD19+ cells but not T cells in the lung at days 14 and 28 after Pneumocystis inoculation. Although anti-CD20 antibody treatment impaired fungal clearance at day 14 postinfection, fungal burden in the lungs was substantially reduced at day 28 in both depleted and control mice in the low-dose group. Subcutaneous anti-CD20 antibody treatment did not alter antigen-specific serum immunoglobulin levels in mice compared with control mice, and there were no significant differences in the numbers of lung gamma interferon-positive (IFN-γ+) CD4+, interleukin 4-positive (IL-4+) CD4+, IL-5+ CD4+, and IL-17A+ CD4+ cells between depleted and control mice after infection. In mice with secondary infection, the lung fungal burden was comparable between depleted and control mice 14 days after reinfection. Low-dose subcutaneous anti-CD20 antibody treatment may delay fungal clearance, but it did not impair the ability of the host to clear Pneumocystis infection, irrespective of primary or secondary infection.IMPORTANCE Anti-CD20 antibody therapy is used for both cancer and autoimmune disease but has been shown to be associated with Pneumocystis pneumonia in humans. This study shows that low-dose subcutaneous anti-CD20 can modulate B cell populations without grossly perturbing fungal immunity against Pneumocystis lung infection.

Subcutaneous anti-CD20 antibody treatment delays gray matter atrophy in human myelin oligodendrocyte glycoprotein-induced EAE mice.

BACKGROUND: The human myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (huMOG-EAE) model, generates B-cell driven demyelination in mice, making it a suitable multiple sclerosis model to study B cell depletion. OBJECTIVES: We investigated the effect of subcutaneous anti-CD20 antibody treatment on huMOG-EAE gray matter (GM) pathology. METHODS: C57Bl/6, 8-week old mice were immunized with 200 huMOG1-125 and treated with 50 µg/mouse of anti-CD20 antibody (n = 16) or isotype control (n = 16). Serial brain volumetric 9.4 T MRI scans was performed at baseline, 1 and 5 wkPI. Disease severity was measured by clinical disability score (CDS) and performance on rotarod test. RESULTS: Anti-CD20 antibody significantly reduced brain volume loss compared with the isotype control across all timepoints longitudinally in the basal ganglia (p = 0.01), isocortex (p = 0.025) and thalamus (p = 0.023). The CDS was reduced significantly with anti-CD20 antibody vs. the isotype control at 3 (p = 0.003) and 4 (p = 0.03) wkPI, while a trend was observed at 5 (p = 0.057) and 6 (p = 0.086) wkPI. Performance on rotarod was also improved significantly at 3 (p = 0.007) and 5 (p = 0.01) wkPI compared with the isotype control. At cellular level, anti-CD20 therapy suppressed the percentage of proliferative nuclear antigen positive microglia in huMOG-EAE isocortex (p = 0.016). Flow cytometry confirmed that anti-CD20 antibody strongly depleted the CD19-expressing B cell fraction in peripheral blood mononuclear cells, reducing it from 39.7% measured in isotype control to 1.59% in anti-CD20 treated mice (p < 0.001). CONCLUSIONS: Anti-CD20 antibody treatment delayed brain tissue neurodegeneration in GM, and showed clinical benefit on measures of disease severity in huMOG-EAE mice.

The Influence of B Cell Depletion Therapy on Naturally Acquired Immunity to Streptococcus pneumoniae.

The anti-CD20 antibody Rituximab to deplete CD20+ B cells is an effective treatment for rheumatoid arthritis and B cell malignancies, but is associated with an increased incidence of respiratory infections. Using mouse models we have investigated the consequences of B cell depletion on natural and acquired humoral immunity to Streptococcus pneumoniae. B cell depletion of naïve C57Bl/6 mice reduced natural IgM recognition of S. pneumoniae, but did not increase susceptibility to S. pneumoniae pneumonia. ELISA and flow cytometry assays demonstrated significantly reduced IgG and IgM recognition of S. pneumoniae in sera from mice treated with B cell depletion prior to S. pneumoniae nasopharyngeal colonization compared to untreated mice. Colonization induced antibody responses to protein rather than capsular antigen, and when measured using a protein array B cell depletion prior to colonization reduced serum levels of IgG to several protein antigens. However, B cell depleted S. pneumoniae colonized mice were still partially protected against both lung infection and septicemia when challenged with S. pneumoniae after reconstitution of their B cells. These data indicate that although B cell depletion markedly impairs antibody recognition of S. pneumoniae in colonized mice, some protective immunity is maintained, perhaps mediated by cellular immunity.

Low-Dose Subcutaneous Anti-CD20 Treatment Depletes Disease Relevant B Cell Subsets and Attenuates Neuroinflammation.

To explore the B cell depleting capacity of a low-dose (20 µg) subcutaneous mouse anti-CD20 antibody treatment on disease-relevant B cell populations within lymph nodes and the spleen. B cell depleting capacity was explored in healthy female C57BL/6 and BALB/c mice; following immune activation in two different mouse models: trinitrophenylated lipopolysaccharide model (thymus-independent response) and dinitrophenyl-keyhole limpet hemocyanin model (thymus-dependent response); and in a chronic neuroinflammation experimental autoimmune encephalomyelitis model. CD20 protein expression on B cell subpopulations was also studied. The subcutaneous anti-CD20 regimen resulted in rapid depletion of B cells in blood, lymph nodes and spleen. Low-dose subcutaneous treatment did not reduce antigen-specific immunoglobulin M and immunoglobulin G titers in all subgroups, and relatively spared splenic marginal zone (MZ) B cells in both T cell dependent and T cell independent B cell immunization models. Analysis of immune compartments during anti-CD20-modulated autoimmune neuroinflammation showed that the maximal B cell depletion was achieved within 2 days of treatment and was highest in the lymph node. Regardless of the tissues analyzed, low-dose subcutaneous treatment was characterized by rapid B cell repletion following treatment cessation. CD20 protein expression was consistent on all B cell subsets in blood, and was more pronounced in germinal center B cells of lymph nodes and MZ B-cells of the spleen. Low-dose subcutaneous anti-CD20 therapy effectively depleted B cells within lymphatic tissues and reduced the severity of neuroinflammation. These data suggest that subcutaneous anti-CD20 therapies can effectively target disease-relevant B cell populations, have shorter repletion kinetics and maintain vaccination responses, thereby achieving autoimmune amelioration without severely impacting immune surveillance functions. Graphical Abstract *p < 0.05; **p < 0.01. CD, cluster of differentiation; DNP-KLH, dinitrophenyl-keyhole limpet hemocyanin; EC50, concentration of a drug that gives half-maximal response; Ig, immunoglobulin; MZ, marginal zone; s.c., subcutaneous; SEM, standard error of mean; TNP-LPS, trinitrophenylatedlipopolysaccharide.

Deficiency of MALT1 paracaspase activity results in unbalanced regulatory and effector T and B cell responses leading to multiorgan inflammation.

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

CD47 fusion protein targets CD172a+ cells in Crohn's disease and dampens the production of IL-1ß and TNF.

In mice, the transfer of CD172a(+) (SIRP-α) dendritic cells (DCs) elicits T cell-driven colitis, whereas treatment with CD47-Fc protein, a CD172a-binding agent, confers protection. The aim of this study was to elucidate the nature and functional properties of human CD172a(+) DCs in chronic intestinal inflammation. Here, we show that CD172a(+)CD11c(+) cells accumulate in the mesenteric lymph nodes (mLNs) and inflamed intestinal mucosa in patients with Crohn's disease (CD). These cells are distinct from resident DCs and may coexpress markers typically associated with monocyte-derived inflammatory DCs such as CD14 and/or DC-SIGN, E-Cadherin, and/or CX3CR1. Spontaneous IL-1ß and TNF production by HLA-DR(+) cells in CD tissues is restricted to those expressing CD172a. An avidity-improved CD47 fusion protein (CD47-Var1) suppresses the release of a wide array of inflammatory cytokines by CD172a(+) cells, which may include HLA-DR(-)CD172a(+) neutrophils, in inflamed colonic explant cultures and impairs the ability of HLA-DR(+)CD172a(+) cells to activate memory Th17 but not Th1 responses in mLNs. In conclusion, targeting CD172a(+) cells may represent novel therapeutic perspectives for patients with CD.

Vav1 GEF activity is required for T cell mediated allograft rejection.

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression.

New pyrazolo[1,5a]pyrimidines as orally active inhibitors of Lck.

A novel series of pyrazolo[1,5a]pyrimidines was optimized to target lymphocyte-specific kinase (Lck). An efficient synthetic route was developed and SAR studies toward activity and selectivity are described, leading to Lck inhibitors with enzymatic, cellular and in vivo potency.

Vav1 couples the T cell receptor to cAMP response element activation via a PKC-dependent pathway.

The transcription factor cAMP-responsive element binding protein (CREB) is a regulator of the expression of several genes important for lymphocyte activation and proliferation. However, the proximal signaling events leading to activation of CREB in T cells upon antigen receptor stimulation remain unknown. Here we identify a role for Vav1 in the activation of the cAMP response element (CRE), the binding site for CREB. T cell receptor (TCR)/CD28 - induced costimulation of Jurkat T cells expressing Vav1 but not a GEF-deficient mutant showed increased CRE activation (7.2+/-2.4 fold over control), whereas Vav1 downregulation by siRNA reduced activation of CRE by 2.6+/-1.3 fold. Inhibition of PKC and MEK but not p38 could reduce Vav1-mediated CRE activation, suggesting that Vav1 transmits TCR and CD28 signals to activation of CRE via PKC and ERK signaling pathways. As a consequence, downregulation of Vav1 impaired the expression of several CRE-containing genes like cyclin D1, INFgamma and IL-2, whereas overexpression of Vav1 enhanced CRE-dependent gene expression. Furthermore, cAMP-induced CRE-dependent transcription and gene expression was also modulated by Vav1, but did not require activation of PKC and the GEF function of Vav1. Our data provide insights into the signal transduction events regulating CRE-mediated gene expression in T cells, which affects T cell development, proliferation and activation. We identify Vav1 as an essential component of TCR-induced CRE activation and gene expression, which underlines the central role for Vav1 as key player for TCR signal transduction and gene expression.

Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1.

The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of Rho GTPases. The structure of the Vav1 DH-PH-CRD/Rac1 complex to 2.6 A resolution reveals a unique intramolecular network of contacts between the Vav1 cysteine-rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav1 DH domain for its intimate association with the Switch II region of Rac1 that is critical for the displacement of the guanine nucleotide. Small angle x-ray scattering (SAXS) studies support this domain arrangement for the complex in solution. Further, mutational analyses confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1's ability to contact a range of Rho GTPases using a novel protein-protein interaction network.

Intracellular Phospho-Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot.

Phospho-site specific antibodies become increasingly available, enabling the study of signaling events by Western blotting (WB) or intracellular flow cytometry (Phospho-Flow). Here we compared data generated by WB or Phospho-Flow regarding the kinetics and degree of phosphorylation of membrane proximal TCR signaling molecules. Phosphorylation events in Jurkat T cells were triggered by anti-CD3 stimulation (OKT3) or by oxidative stress (H(2)O(2)) and were analyzed by Phospho-Flow or WB. Both techniques showed that OKT3- or H(2)O(2)-induced, transient phosphorylation of ZAP70 or LAT was dependent on functional Lck. Phospho-Flow data revealed differences in the kinetics and the degree of H(2)O(2)- or OKT3-mediated protein phosphorylation compared with WB data. In addition, using Phospho-Flow we discovered that H(2)O(2)-induced phosphorylation of TCR signaling proteins was inhibited by small molecular weight kinase inhibitors far more potently than OKT3-triggered protein phosphorylation, despite a superior induction of phosphorylation by H(2)O(2). This finding was confirmed by WB. Interestingly, we identified by Phospho-Flow that, in P116 Jurkat cells lacking ZAP70 protein expression, H(2)O(2) potently triggered the phosphorylation of ZAP70 residues Y493 and Y292 but not Y319. The phosphorylation of these ZAP70 tyrosine residues cells was blocked by an Lck inhibitor, suggesting the existence of an Lck-coupled truncated ZAP70 protein or a novel isoform of ZAP70 in P116 cells. Phospho-Flow is a largely quantitative technology with excellent throughput, highly suited in studying the function or inhibition of TCR signaling pathways and allowing the detection of novel pathway insights. It can serve as a good complement to Western blot analysis.

Strongly reduced alloreactivity and long-term survival times of cardiac allografts in Vav1- and Vav1/Vav2-knockout mice.

Vav proteins mediate T- and B-cell activation by functioning as GTP/GDP exchange factors for small GTPases. We have studied the role of Vav1 and Vav2 in allogeneic T-cell activation, antibody responses and allograft rejection. Alloantigen-induced proliferation of T cells from Vav1- and Vav1/Vav2-knockout (ko) mice was decreased by >90% in a mixed lymphocyte reaction. In whole-blood cultures, Vav deficiency led to markedly impaired T- and B-cell activation. Expansion of Vav1- or Vav1/Vav2-ko T cells (C57BL/6) was reduced after transfer into severe combined immune deficiency/beige recipient mice (BALB/c). After priming with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin, T-cell-dependent anti-DNP IgM and IgG antibody levels were normal in Vav1-ko mice but undetectable in Vav1/Vav2-ko mice. The median survival time of BALB/c cardiac allografts transplanted into C57BL/6 Vav1-ko mice (n = 13) or Vav1/Vav2-ko mice (n = 5) was >100 and >77 days, compared with 8-9 days in the corresponding wild-type mice. Vav1/Vav2-ko mice with <100 days graft survival developed bacterial skin infections and were prematurely killed with beating cardiac allograft. Long-term surviving transplants of single and double ko mice showed mild cellular interstitial rejection and mild to severe vascular remodeling. In conclusion, our studies show for the first time that the absence of Vav1 and Vav1/Vav2 in ko mice strongly reduces alloreactivity and results in long-term allograft survival, whereas antibody responses were only affected in Vav1/Vav2 ko mice.

Remedial strategies in structural proteomics: expression, purification, and crystallization of the Vav1/Rac1 complex.

The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here, we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.

The multi-ligand somatostatin analogue SOM230 inhibits ACTH secretion by cultured human corticotroph adenomas via somatostatin receptor type 5.

OBJECTIVE: Currently, there is no effective medical treatment for patients with pituitary-dependent Cushing's disease. A novel somatostatin (SS) analogue, named SOM230, with high binding affinity to SS receptor subtypes sst(1), sst(2), sst(3) and sst(5) was recently introduced. We compared the in vitro effects of the sst(2)-preferring SS analogue octreotide (OCT) and the multi-ligand SOM230 on ACTH release by human and mouse corticotroph tumour cells. METHODS: By quantitative RT-PCR the sst subtype expression level was determined in human corticotroph adenomas. In vitro, the inhibitory effect of OCT and SOM230 on ACTH release by dispersed human corticotroph adenoma cells and mouse AtT20 corticotroph adenoma cells was determined. In addition, the influence of dexamethasone on the responsiveness to OCT and SOM230 was studied. RESULTS: Corticotroph adenomas expressed predominantly sst(5) mRNA (six out of six adenomas), whereas sst(2) mRNA expression was detected at significantly lower levels. In a 72 h incubation with 10 nmol/l SOM230, ACTH release was inhibited in three out of five cultures (range -30 to -40%). Ten nmol/l OCT slightly inhibited ACTH release in only one of five cultures (- 28%). In AtT20 cells, expressing sst(2), sst(3) and sst(5), SOM230 inhibited ACTH secretion with high potency (IC(50) 0.2 nmol/l). Dexamethasone (10 nmol/l) pre-treatment did not influence the sensitivity of the cells to the inhibitory effect of SOM230, suggesting that sst(5) is relatively resistant to negative control by glucocorticoids. CONCLUSIONS: The selective expression of sst(5) receptors in corticotroph adenomas and the preferential inhibition of ACTH release by human corticotroph adenoma cells by SOM230 in vitro, suggest that SOM230 may have potential in the treatment of patients with pituitary-dependent Cushing's disease.

The novel somatostatin ligand (SOM230) regulates human and rat anterior pituitary hormone secretion.

Currently available somatostatin analogs predominantly bind to the somatostatin receptor subtype (SSTR)2 subtype, and control GH and IGF-I secretion in approximately 65% of patients with acromegaly, their efficacy relating to receptor density and subtype expression. SOM230 is a somatostatin ligand with high affinity to four SSTR subtypes. In primary cultures of rat pituicytes, SOM230 dose-dependently inhibited GH release (P = 0.002) with an IC50 of 1.2 nM. Ten nanomoles SOM230 inhibited GH and TSH release by 40 +/- 7% (P < 0.001) and 47 +/- 21% (P = 0.09), respectively. No effect of SOM230 was observed on prolactin (PRL) or LH release. In cultures of human fetal pituitary cells, SOM230 inhibited GH secretion by 42 +/- 9% (P = 0.002) but had no effect on TSH release. SOM230 inhibited GH release from GH-secreting adenoma cultures by 34 +/- 8% (P = 0.002), PRL by 35 +/- 4% from PRL-secreting adenomas (P = 0.01), and alpha-subunit secretion from nonfunctioning pituitary adenomas by 46 +/- 18% (P = 0.34). In contrast, octreotide inhibited GH, PRL, and alpha-subunit from the respective adenoma by 18 +/- 12 (P = 0.39), 22 +/- 4 (P = 0.04), and 20 +/- 10% (P = 0.34). In all culture systems, no significant difference in the inhibitory action of SOM230, octreotide, and somatostatin 14 on hormone release was observed. SOM230, similar to somatostatin, has high-affinity binding to SSTR1, 2, 3, and 5 and, in keeping with this, has an equivalent inhibitory effect on pituitary hormone secretion. As a consequence of its broader binding profile, SOM230 is likely to find clinical utility in treating tumors resistant to SSTR-2-preferential analogs.

The novel somatostatin analog SOM230 is a potent inhibitor of hormone release by growth hormone- and prolactin-secreting pituitary adenomas in vitro.

To determine the inhibitory profile of the novel somatostatin (SRIF) analog SOM230 with broad SRIF receptor binding, we compared the in vitro effects of SOM230, octreotide (OCT), and SRIF-14 on hormone release by cultures of different types of secreting pituitary adenomas. OCT (10 nM) significantly inhibited GH release in seven of nine GH-secreting pituitary adenoma cultures (range, -26 to -73%), SOM230 (10 nM) in eight of nine cultures (range, -22 to -68%), and SRIF-14 (10 nM) in six of six cultures (range, -30 to -75%). The sst analysis showed predominant but variable levels of somatostatin receptor (sst)(2) and sst(5) mRNA expression. In one culture completely resistant to OCT, SOM230 and SRIF-14 significantly inhibited GH release in a dose-dependent manner with an IC(50) value in the low nanomolar range. In the other cultures, SOM230 showed a lower potency of GH release inhibition (IC(50), 0.5 nM), compared with OCT (IC(50), 0.02 nM) and SRIF-14 (IC(50), 0.02 nM). A positive correlation was found between sst(2) but not sst(5) mRNA levels in the adenoma cells and the inhibitory potency of OCT on GH release in vivo and in vitro, and the effects of SOM230 and SRIF-14 in vitro. In three prolactinoma cultures, 10 nM OCT weakly inhibited prolactin (PRL) release in only one (-28%), whereas 10 nM SOM230 significantly inhibited PRL release in three of three cultures (-23, -51, and -64.0%). The inhibition of PRL release by SOM230 was related to the expression level of sst(5) but not sst(2) mRNA. Several conclusions were reached. First, SOM230 has a broad profile of inhibition of tumoral pituitary hormone release in the low nanomolar range, probably mediated via both sst(2) and sst(5) receptors. The higher number of responders of GH-secreting pituitary adenoma cultures to SOM230, compared with OCT, suggest that SOM230 has the potency to increase the number of acromegalic patients which can be biochemically controlled. Second, compared with OCT, SOM230 is more potent in inhibiting PRL release by mixed GH/PRL-secreting adenoma and prolactinoma cells.

A single-dose comparison of the acute effects between the new somatostatin analog SOM230 and octreotide in acromegalic patients.

Treatment with the somatostatin receptor (sst) subtype 2 predominant analogs octreotide and lanreotide induces clinical and biochemical cure in approximately 65% of acromegalic patients. GH-secreting pituitary adenomas, which are not controlled, also express sst(5). We compared the acute effects of octreotide and SOM230, a new somatostatin analog with high affinity for sst(1,2,3,5) on hormone release in acromegalic patients. In a single-dose, proof-of-concept study, 100 microg octreotide and 100 and 250 microg SOM230 were given s.c. to 12 patients with active acromegaly. Doses of 100 and 250 microg SOM230 dose-dependently suppressed GH levels from 2-8 h after administration (-38 +/- 7.7 vs. -61 +/- 6.7%, respectively; P < 0.01). A comparable suppression of GH levels by octreotide and 250 microg SOM230 was observed in eight patients (-65 +/- 7 vs. -72 +/- 7%, respectively). In three patients, the acute GH-lowering effect of 250 microg SOM230 was significantly superior to that of octreotide (-70 +/- 2 vs. -17 +/- 15%, respectively; P < 0.01). In one patient, the GH-lowering effect of octreotide was better than that of SOM230. Tolerability for SOM230 was good. Glucose levels were initially slightly elevated after octreotide and SOM230, compared with control day, whereas insulin levels were only significantly suppressed by octreotide. We conclude that SOM230 is an effective GH-lowering drug in acromegalic patients with the potential to increase the number of patients controlled during long-term medical treatment.

Drug design at peptide receptors: somatostatin receptor ligands.

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 affinitty 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., [125]LTT-SRIF28' [l25I]CGP23996, [125]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 SRIF2receptors 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 ith 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.