Combination of a proteomics approach and reengineering of meso scale network models for prediction of mode-of-action for tyrosine kinase inhibitors.

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.

Receptor-targeted therapy of human experimental urinary bladder cancers with cytotoxic LH-RH analog AN-152 [AEZS- 108].

Many bladder cancers progress to invasion with poor prognosis; new therapeutic methods are needed. We developed a cytotoxic LH-RH analog, AN-152 (AEZS-108) containing doxorubicin (DOX), for targeted therapy of cancers expressing LHRH receptors. We investigated the expression of LH-RH receptors in clinical bladder cancers and in HT-1376, J82, RT-4 and HT-1197 human bladder cancer lines. The effect of analog, AN-152, on growth of these tumor lines xenografted into nude mice was analyzed. Using molecular and functional assays, we also evaluated the differences between the effects of AN-152, and DOX alone. We demonstrated the expression of LH-RH receptors on 18 clinical bladder cancers by immunohistochemistry and on four human urinary bladder cancer lines HT-1376, J82, RT-4 and HT-1197 by Western blotting and binding assays. AN-152 powerfully inhibited growth of these bladder cancers in nude mice. AN-152 exerted greater effects than DOX and was less toxic. DOX activated strong multidrug resistance mechanisms in RT-4 and HT-1197 cancers, while AN-152 had no or less such effect. PCR assays and in vitro studies revealed differences in the action of AN-152 and DOX on the expression of genes involved in apoptosis. These results suggest that targeted cytotoxic LH-RH analog, AN-152 (AEZS- 108), should be examined for treatment of patients with LH-RH receptor positive invasive bladder cancers.

Targeting aurora kinases with danusertib (PHA-739358) inhibits growth of liver metastases from gastroenteropancreatic neuroendocrine tumors in an orthotopic xenograft model.

PURPOSE: Aurora kinases play a crucial role in cell-cycle control. Uncontrolled expression of aurora kinases causes aneuploidy and tumor growth. As conservative treatment options for advanced gastroenteropancreatic neuroendocrine tumors (GEP-NET) are disappointing, aurora kinases may be an interesting target for novel therapeutic strategies. EXPERIMENTAL DESIGN: Human GEP-NETs were tested for aurora kinase expression. The efficacy of the new aurora kinase inhibitor danusertib was evaluated in two human GEP-NET cell lines (BON1 and QGP) in vitro and in vivo. RESULTS: The majority of ten insulinomas and all 33 nonfunctional pancreatic or midgut GEP-NETs expressed aurora A despite a mostly high degree of cell differentiation. Both human GEP-NET cell lines expressed aurora kinase A and B, and high Ser10 phosphorylation of histone H3 revealed increased aurora B activity. Remarkably, danusertib led to cell-cycle arrest and completely inhibited cell proliferation of the GEP-NET cells in vitro. Decreased phosphorylation of histone H3 indicated effective aurora B inhibition. In a subcutaneous murine xenograft model, danusertib significantly reduced tumor growth in vivo compared with controls or mice treated with streptozotocine/5-fluorouracil. As a consequence, decreased levels of tumor marker chromogranin A were found in mouse serum samples. In a newly developed orthotopic model for GEP-NET liver metastases by intrasplenic tumor cell transplantation, dynamic MRI proved significant growth inhibition of BON1- and QGP-derived liver metastases. CONCLUSIONS: These results show that danusertib may impose a new therapeutic strategy for aurora kinase expressing metastasized GEP-NETs.

Multi tyrosine kinase inhibitor dasatinib as novel cause of severe pre-capillary pulmonary hypertension?

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disease with poor prognosis. Encouraging efforts have been made to target the main vasoproliferative aspects of the disease. Promising emerging therapeutics are tyrosine kinase inhibitors such as imatinib. CASE PRESENTATION: Here, we discuss the relevance of previously published cases and add another well-characterised patient who developed pre-capillary PH under long-term therapy with the multi-tyrosine kinase inhibitor dasatinib approved for therapy of chronic myeloic leukaemia (CML) and Philadelphia chromosome positive acute lymphocytic leukaemia (mean time of all patients on dasatinib: 26 months). Hence, we discuss the possibility of dasatinib itself causing PH after long-term therapy and turn specialist's attention to this possible severe side effect.At present, the true incidence of dasatinib-associated PH remains illusive and systematic data regarding haemodynamics are missing. CONCLUSION: We therefore recommend systematic screening of dasatinib-treated patients for pulmonary hypertension and subsequent collection of haemodynamic data.

Abcg2 overexpression represents a novel mechanism for acquired resistance to the multi-kinase inhibitor Danusertib in BCR-ABL-positive cells in vitro.

The success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance in vitro. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance in vitro, raising hope that this drug combination may also achieve more durable disease control in vivo.

Bosutinib.

Bosutinib (SKI-606) is a 7-alkoxy-3-quinolinecarbonitrile, which functions as a dual inhibitor of Src and Abl kinases. In biochemical and proliferation assays, the compound was shown to be active against src family kinases and Bcr-Abl at IC50s of 100 and 90 nM, respectively. The bcr-abl fusion gene product, a consecutively activated tyrosine kinase, which is crucial for the development of chronic myeloid leukaemia (CML), is highly sensitive to bosutinib. Interestingly, distinctly lower concentrations of the dual src/abl inhibitor are required to ablate Bcr-Abl phosphorylation when compared to first-generation tyrosine kinase inhibitor imatinib (IM). Bosutinib is a potent inhibitor of CML cell proliferation in vitro and in vivo experiments and has demonstrated promising harbouring results in CML patients resistance or intolerance to IM in ongoing phase I/II clinical trials. Remarkably, bosutinib has been found to be capable of overcoming the majority of IM-resistant bcr-abl mutations. A randomised open label phase III clinical study to compare the efficacy of bosutinib and IM in first-line therapy of Ph+ chronic phase (CP) CML has recently been initiated. In a phase I/II clinical study with subjects suffering from advanced stages of solid tumours, long-term responses have also been reported. In conclusion, Bosutinib is a promising novel small molecule inhibitor for targeted therapy of CML and solid tumours.

Aurora kinase inhibitor PHA-739358 suppresses growth of hepatocellular carcinoma in vitro and in a xenograft mouse model.

Patients with advanced stages of hepatocellular carcinoma (HCC) face a poor prognosis. Although encouraging clinical results have been obtained with multikinase inhibitor sorafenib, the development of improved therapeutic strategies for HCC remains an urgent goal. Aurora kinases are key regulators of the cell cycle, and their uncontrolled expression promotes aneuploidy and tumor development. In tissue microarray analyses, we detected aurora-A kinase expression in all of the examined 93 human HCC samples, whereas aurora-B kinase expression levels significantly correlated with the proliferation index of HCCs. In addition, two human HCC cell lines (Huh-7 and HepG2) were tested positive for aurora-A and -B and revealed Ser10 phosphorylation of histone H3, indicating an increased aurora-B kinase activity. The antiproliferative features of a novel aurora kinase inhibitor, PHA-739358, currently under investigation in phase 2 clinical trials for other solid tumors, were examined in vitro and in vivo. At concentrations exceeding 50 nM, PHA-739358 completely suppressed tumor cell proliferation in cell culture experiments and strongly decreased histone H3 phosphorylation. Cell cycle inhibition and endoreduplication were observed at 50 nM, whereas higher concentrations led to a complete G(2)/M-phase arrest. In vivo, administration of PHA-739358 resulted in significant tumor growth inhibition at a well-tolerated dose. In combination with sorafenib, additive effects were observed. Remarkably, when tumors restarted to grow under sorafenib monotherapy, subsequent treatment with PHA-739358 induced tumor shrinkage by up to 81%. Thus, targeting aurora kinases with PHA-739358 is a promising therapeutic strategy administered alone or in combination with sorafenib for patients with advanced stages of HCC.

Telomeres and telomerase in chronic myeloid leukaemia: impact for pathogenesis, disease progression and targeted therapy.

Telomeres are specialized structures localized at the end of human chromosomes. Due to the end replication problem, each cell division results in a loss of telomeric repeats in normal somatic cells. In germ line and stem cells, the multicomponent enzyme telomerase maintains the length of telomere repeats. However, elevated telomerase activity has also been reported in the majority of solid tumours as well as in acute and chronic leukaemia. Chronic myeloid leukaemia (CML) serves as a model disease to study telomere biology in clonal myeloproliferative disorders. In CML, telomere shortening correlates with disease stage, duration of chronic phase (CP), prognosis measured by the Hasford risk score and the response to disease-modifying therapeutics such as the tyrosine kinase inhibitor Imatinib. In addition, telomerase activity (TA) is already increased in CP CML and further upregulated with disease progression to accelerated phase and blast crisis (BC). Furthermore, a correlation of TA with increased genetic instability as well as a shorter survival of the patients has been reported. Here, we review the current state of knowledge of the role of telomere and telomerase biology in CML and discuss the possible impact of novel treatment approaches.

Bosutinib: a dual SRC/ABL kinase inhibitor for the treatment of chronic myeloid leukemia.

The tyrosine kinase inhibitor imatinib mesylate (IM) set new standards in the treatment of chronic myeloid leukemia (CML). However, emergence of resistance to IM became a major therapeutic challenge. Bosutinib (SKI-606), a 7-alkoxy-3-quinolinecarbonitrile, functions as a dual inhibitor of SRC and ABL kinases, and preclinical studies demonstrated a high antiproliferative activity in human and murine CML cell lines. In ongoing Phase I/II clinical trials, bosutinib yielded promising results revealing high clinical efficacy, good tolerability and reduced toxicity in IM-resistant or -intolerant CML patients. In this article, we provide an overview on the mechanism of action, and the preclinical and currently available clinical data for bosutinib. Owing to its favorable toxicity profile and its high antileukemic activity, bosutinib is a promising novel treatment option for patients with CML. A recently initiated, randomized open-label Phase III clinical study will clarify its role in first-line therapy of Philadelphia chromosome-positive chronic-phase CML.

BRAFV600E mutations in malignant melanoma are associated with increased expressions of BAALC.

UNLABELLED: BACHGROUND: Activating BRAF mutations are present in approximately 50% of melanomas. Although different downstream target genes of the most common mutant V600E have been identified, the contribution of activating BRAF mutations to malignant transformation needs further clarification. METHODS: Microarray gene analysis was performed for human melanoma cell lines harboring BRAFV600E mutations in comparison to cell lines without this mutation. RESULTS: This analysis revealed a more than two fold down-regulation of 43 and an increase of 39 gene products. BAALC (Brain and acute Leukaemia, cytoplasmatic) was most prominently regulated, since it was up-regulated in mutated cell lines by a mean of 11.45. Real time PCR analyses with RNA from melanoma cell lines (n = 30) confirmed the BRAF-activation dependent up-regulation of BAALC. CONCLUSION: BAALC, which has been associated with cell dedifferentiation and migration, may function as a downstream effector of activating BRAF mutations during melanomagenesis.

PHA-680626 exhibits anti-proliferative and pro-apoptotic activity on Imatinib-resistant chronic myeloid leukemia cell lines and primary CD34+ cells by inhibition of both Bcr-Abl tyrosine kinase and Aurora kinases.

Emergence of resistance to Imatinib complicates the treatment of chronic myeloid leukemia (CML). Second-generation Bcr-Abl inhibitors are capable to overcome resistance mediated by most mutations except T315I. As this mutation is causative for approximately 20% of clinically observed resistances, the need for novel treatment strategies becomes obvious. Here, we report on a novel kinase inhibitor PHA-680626 exhibiting strong inhibitory activity on both Bcr-Abl and Aurora kinases. Significant anti-proliferative and pro-apoptotic effects were observed in human BCR-ABL positive cell lines and murine BaF3 cells ectopically expressing wt BCR-ABL or the Imatinib-resistant BCR-ABL mutants M351T, E255K and, T315I. Treatment with PHA-680626 decreased phosphorylation of CrkL and histone H3. As CrkL represents a typical downstream target of Bcr-Abl while histone H3 phosphorylation is an indicator for Aurora kinase B activity, these findings indicate that effects of PHA-680626 are mediated via inhibition of both pathways. Moreover, high anti-proliferative activity of PHA-680626 was observed in primary CD34+ cells derived from CML patients at diagnosis or in blast crisis as well as from an individual harbouring the T315I mutation. Thus, both Bcr-Abl and Aurora kinase inhibition contribute to the efficacy of PHA-680626 against Imatinib-resistant BCR-ABL positive leukemias, particularly those harbouring the T315I mutation.

Simultaneous targeting of Aurora kinases and Bcr-Abl kinase by the small molecule inhibitor PHA-739358 is effective against imatinib-resistant BCR-ABL mutations including T315I.

The emergence of resistance to imatinib (IM) mediated by mutations in the BCR-ABL domain has become a major challenge in the treatment of chronic myeloid leukemia (CML). Here, we report on studies performed with a novel small molecule inhibitor, PHA-739358, which selectively targets Bcr-Abl and Aurora kinases A to C. PHA-739358 exhibits strong antiproliferative and proapoptotic activity against a broad panel of human BCR-ABL-positive and -negative cell lines and against murine BaF3 cells ectopically expressing wild-type (wt) or IM-resistant BCR-ABL mutants, including T315I. Pharmacologic synergism of IM and PHA-739358 was observed in leukemia cell lines with subtotal resistance to IM. Treatment with PHA-739358 significantly decreased phosphorylation of histone H3, a marker of Aurora B activity and of CrkL, a downstream target of Bcr-Abl, suggesting that PHA-739358 acts via combined inhibition of Bcr-Abl and Aurora kinases. Moreover, strong antiproliferative effects of PHA-739358 were observed in CD34(+) cells derived from untreated CML patients and from IM-resistant individuals in chronic phase or blast crisis, including those harboring the T315I mutation. Thus, PHA-739358 represents a promising new strategy for treatment of IM-resistant BCR-ABL-positive leukemias, including those harboring the T315I mutation. Clinical trials investigating this compound in IM-resistant CML have recently been initiated.

Impact of the CCR5 gene polymorphism on the survival of metastatic melanoma patients receiving immunotherapy.

PURPOSE: Chemokines influence both tumor progression and anti-tumor immune response. A 32-bp-deletion polymorphism in the chemokine receptor 5 gene (CCR5Delta32) has been shown to result in a non-functional protein. This study was aimed at evaluating the potential impact of this gene polymorphism on disease progression and treatment outcome in patients with melanoma. PATIENTS AND METHODS: CCR5 genotyping was performed by PCR on DNA extracted from serum samples of 782 cutaneous melanoma patients with known disease history and long-term clinical follow-up. Genotypes were correlated with patient survival and types of treatment. RESULTS: Of 782 melanoma patients, 90 (11.5%) were heterozygous and 12 (1.5%) were homozygous for CCR5Delta32. Analyzing the complete cohort, the disease-specific survival from date of primary diagnosis was not influenced by CCR5 status. Similarly, no significant impact could be detected on the treatment outcome of stage III patients. In 139 stage IV patients receiving immunotherapy, CCR5Delta32 was associated with a decreased survival compared to patients not carrying the deletion (median 12.5 vs. 20.3 months, P = 0.029). Multivariate analysis revealed the CCR5 genotype as an independent factor impacting disease-specific survival in this patient population (P = 0.002), followed by gender (P = 0.019) and pathological classification of the primary (pT; P = 0.022). CONCLUSION: The presence of the CCR5Delta32 polymorphism in patients with stage IV melanoma results in a decreased survival following immunotherapy and may help to select patients less likely to benefit from this type of treatment.

Therapy of ovarian cancers with targeted cytotoxic analogs of bombesin, somatostatin, and luteinizing hormone-releasing hormone and their combinations.

The aim of this study was to investigate the effect of treatment of experimental ovarian cancers with targeted cytotoxic analogs as single compounds and in combination. Targeted cytotoxic analogs of bombesin (AN-215), somatostatin (AN-238), and luteinizing hormone-releasing hormone (AN-207) consisted of 2-pyrrolinodoxorubicin (AN-201) linked to the respective peptide carrier. AN-238 at 200 nmol/kg significantly inhibited growth of UCI-107, ES-2 and OV-1063 ovarian cancers. AN-215 alone at 200 nmol/kg and its combination with AN-238 at one-half of the dose were also able to inhibit the growth of UCI-107 tumors. A combination of AN-238 with AN-207at 50% of the dose strongly suppressed the proliferation of ES-2 and OV-1063 ovarian tumors. Cytotoxic radical AN-201 was toxic and had no significant effect on tumor growth. In contrast, the toxicity of the conjugated peptide analogs was low. Because ovarian cancers tend to acquire chemoresistance, we used real-time PCR to measure the mRNA expression of multidrug resistance protein 1, multidrug resistance-related protein 1, and breast cancer resistance protein after treatment. Low or no induction of multidrug resistance protein 1, multidrug resistance-related protein, and breast cancer resistance protein occurred after treatment with AN-238, AN-215, and the combination of AN-238 with AN-207 or AN-215. These results demonstrate that a therapy with cytotoxic analogs such as single agents and combinations is effective and nontoxic. Our work suggests that cytotoxic peptide analogs of luteinizing hormone-releasing hormone, somatostatin, and bombesin could be used for the therapy of ovarian cancers, considering the lack of induction of chemoresistance.

Effective therapy of experimental human malignant melanomas with a targeted cytotoxic somatostatin analogue without induction of multi-drug resistance proteins.

Malignant melanomas are characterized by a high intrinsic resistance to chemotherapy. Multiple drug resistance (MDR) can be mediated by transport proteins such as MDR-1, multidrug resistance-associated protein (MRP) or lung resistance protein (LRP). The cytotoxic analogue of somatostatin AN-238 consisting of 2-pyrrolinodoxorubicin (AN-201) linked to a somatostatin analogue RC-121 binds to receptors for somatostatin and is targeted to tumors expressing these receptors. We evaluated the expression of somatostatin receptors on human malignant melanoma tumor lines MRI-H255 and MRI-H187 and examined the effects of the targeted analogue AN-238 and its cytotoxic radical AN-201 on growth of these tumors in nude mice. We also studied the effects of AN-238 and AN-201 on the expression of MDR-1, MRP-1 and LRP by real-time PCR. AN-238 inhibited the growth of MRI-H255 and MRI-H187 tumors while AN-201 was ineffective. Blockade of somatostatin receptors by somatostatin analogue RC-121 abolished the effects of AN-238. Targeted therapy with AN-238 did not produce an induction of mRNA of MDR-1, MRP-1 or LRP. Our findings show that targeted chemotherapy with cytotoxic somatostatin analogue AN-238 inhibits the growth of malignant melanomas. AN-238 could provide a novel treatment approach for advanced malignant melanomas.

Targeted cytotoxic bombesin analog AN-215 effectively inhibits experimental human breast cancers with a low induction of multi-drug resistance proteins.

The cytotoxic analog of bombesin (BN)/gastrin releasing peptide (GRP) AN-215 consisting of 2-pyrrolinodoxorubicin (AN-201), a superactive derivative of doxorubicin linked to a bombesin analog carrier, displays a high affinity to BN/GRP receptors and can be targeted to tumors that express these receptors. We evaluated the antitumor effect and the toxicity of AN-215 in 5 human breast cancer cell lines xenografted into nude mice. In addition, we measured the mRNA expression of multi drug resistance protein 1 (MDR-1), multi drug resistance related protein 1 (MRP-1) and breast cancer resistance protein (BCRP) by real-time PCR analysis after treatment with AN-215. All five cell lines expressed BN/GRP receptors, and AN-215 significantly (P < 0.05) inhibited tumor growth in all models, while its cytotoxic radical AN-201 had no significant effect in four models. In MX-1 tumors, AN-201 had a significantly weaker antitumor effect than AN-215. The effect of AN-215 was nullified by a blockade of BN/GRP receptors with a bombesin antagonist. Low or no induction of MDR-1, MRP-1 and BCRP occurred after treatment with AN-215. In conclusion, targeted chemotherapy with the cytotoxic BN/GRP analog AN-215 strongly inhibits breast cancers that express BN/GRP receptors and might provide a new treatment modality for mammary carcinoma.

Targeted chemotherapy with cytotoxic bombesin analogue AN-215 can overcome chemoresistance in experimental renal cell carcinomas.

BACKGROUND: Multidrug resistance (MDR) mediated by membrane transporters, such as P-glycoprotein (MDR-1) and MDR-associated protein (MRP), remains a challenge in the therapy of renal cell carcinoma (RCC). Chemotherapy targeted to hormone receptors may provide a new approach to overcome chemoresistance. The cytotoxic analogue of bombesin/gastrin-releasing peptide (GRP), AN-215, consists of a superactive derivative of doxorubicin, AN-201, which is linked to a bombesin analogue carrier: RC-3094. METHODS: The authors examined the expression of bombesin/GRP receptors in 3 human RCC cell lines (A-498, ACHN. and 786-0) by using reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis and radioligand-binding assays. They also evaluated the effects of AN-215 and its cytotoxic radical AN-201 in the same RCC models in vivo, and they studied the effects of AN-215 and AN-201 on the expression levels of MDR-1 and subtype 1 of MRP (MRP-1) by using real-time PCR. RESULTS: A N-215 significantly (P < 0.05) inhibited the growth of A-498, ACHN, and 786-0 RCC xenografted into nude mice by 59.2-67.6%, whereas the cytotoxic radical AN-201 alone had no significant antitumor effects. The efficacy of AN-215 was independent of the expression patterns of MDR-1 and MRP-1 in these RCC cell lines. The induction of MDR-1 by AN-215 was similar (Experiment 2) or weaker (Experiment 1) compared with AN-201. Both AN-215 and AN-201 caused only a minor induction of MRP-1. CONCLUSIONS: The current findings indicated that targeted chemotherapy with cytotoxic bombesin/GRP analogue AN-215 can inhibit the growth of RCC, providing a new treatment modality for patients with advanced RCC.

Receptors for luteinizing hormone releasing hormone (LHRH) expressed in human non-Hodgkin's lymphomas can be targeted for therapy with the cytotoxic LHRH analogue AN-207.

We determined by immunohistochemistry the presence of LHRH receptors in surgical specimens of human non-Hodgkin's lymphomas (NHL) and investigated the expression of LHRH receptors in two human NHL cell lines, RL and HT by RT-PCR, Western blot and radioligand binding studies. In in vivo experiments with nude mice bearing tumours of these NHL cell lines, the efficacy of cytotoxic LHRH analogue AN-207 and its cytotoxic radical AN-201 was examined. LHRH receptors were detected in 94.1% of the human NHL specimens and in both NHL cell lines. AN-207 significantly (P < 0.01) inhibited the growth of RL and HT tumours, while the non-targeted AN-201 had no effects. Blockade of the LHRH receptors with an excess of LHRH agonist Decapeptyl suppressed the antitumour effects of AN-207. Our findings indicate that LHRH receptors expressed in a high percentage of human NHL specimens can be used for effective targeted therapy with the cytotoxic LHRH analogue AN-207.

Receptors for luteinizing hormone releasing hormone expressed on human renal cell carcinomas can be used for targeted chemotherapy with cytotoxic luteinizing hormone releasing hormone analogues.

PURPOSE: To determine the expression of luteinizing hormone releasing hormone (LHRH) receptors in specimens and cell lines of human renal cell carcinoma (RCC) and to evaluate the antitumor efficacy of targeted therapy with a cytotoxic analogue of LHRH, AN-207, in vivo. AN-207, consisting of [D-Lys(6)] LHRH linked to a cytotoxic radical, 2-pyrrolinodoxorubicin (AN-201), binds with high affinity to LHRH receptors and can be targeted to tumors expressing these receptors. EXPERIMENTAL DESIGN: The expression of LHRH receptors was investigated in 28 surgically removed specimens of human renal cell carcinoma (RCC) by immunohistochemistry and in three human RCC cell lines A-498, ACHN, and 786-0 by radioreceptor assays, Western immunoblotting, and reverse transcription-PCR analysis. Antitumor efficacy of AN-207 was examined in experimental models of these cell lines. RESULTS: Positive staining for LHRH receptors was found in all (28 of 28) of the examined human RCC specimens. mRNA for LHRH receptor, receptor protein, and LHRH binding sites were detected in all three cell lines. AN-207 significantly (P < 0.05) inhibited the growth of A-498, ACHN, and 786-0 xenografts in vivo producing a 67.8% to 73.8% decrease in tumor volume and a 62.2% to 77.3% reduction in tumor weight. Nontargeted cytotoxic radical AN-201 had no significant antitumor effects. Blockade of LHRH receptors by an excess of LHRH agonist Decapeptyl suppressed tumor inhibitory effects of AN-207. CONCLUSIONS: Our findings indicate that LHRH receptors are expressed in human RCC specimens and can be used for targeted chemotherapy with cytotoxic LHRH analogues.

Experimental therapy of human endometrial cancers with a targeted cytotoxic bombesin analog AN-215: low induction of multidrug resistance proteins.

In this study we have investigated the efficacy and toxicity of targeted cytotoxic bombesin (BN) analog AN-215 and its effects on the expression of three multidrug resistance proteins in experimental human endometrial cancers. Nude mice bearing HEC-1A, RL-95-2 and AN3CA tumours were treated with AN-215 and its cytotoxic radical (AN-201). The BN receptor expression in tumours was followed by RT-PCR analysis and radioligand binding assays. Expression of drug resistance proteins MDR-1, MRP-1 and BCRP were measured by realtime PCR. AN-215 significantly (P<0.05) inhibited the growth of HEC-1A, RL-95-2 and AN3CA tumours while AN-201 was ineffective. The expression of BN receptors was demonstrated in all three tumour models. AN-215 caused a lower induction of MDR-1 in HEC-1A and RL-95-2 cancers than AN-201. MRP-1 and BCRP were not induced by AN-215 or AN-201. Thus, targeted chemotherapy with AN-215 powerfully inhibits the growth of human BN receptor-positive endometrial cancers.