SURPASS-ET: phase III study of ropeginterferon alfa-2b versus anagrelide as second-line therapy in essential thrombocythemia.

Patients diagnosed with high-risk essential thrombocythemia (ET) have limited treatment options to reduce the risk of thrombosis and lessen the progression of the disease by targeting the molecular source. Hydroxyurea is the recommended treatment, but many patients experience resistance or intolerance. Anagrelide is an approved second-line option for ET, but concerns of a higher frequency of disease transformation may affect its role as a suitable long-term option. Interferons have been evaluated in myeloproliferative neoplasms for over 30 years, but early formulations had safety and tolerability issues. SURPASS-ET (NCT04285086) is a phase III, open-label, multicenter, global, randomized, active-controlled trial that will evaluate the safety, efficacy, tolerability and pharmacokinetics of ropeginterferon alfa-2b compared with anagrelide as second-line therapy in high-risk ET.

Essential thrombocythemia (ET) is a condition characterized by having more platelets than normal. The high number of platelets increases the risk of a life-threatening blood clot and/or bleeding. Patients with ET and at a high risk for these events are usually treated first with hydroxyurea (HU), but some patients do not respond properly or may develop significant side effects. Anagrelide is an approved medication used in patients who do not respond to HU. Ropeginterferon alfa-2b is a disease-specific, long-acting interferon with a good safety profile approved in polycythemia vera, another type of myeloproliferative neoplasm. The SURPASS-ET clinical trial will evaluate the safety, efficacy, tolerability and pharmacokinetics of ropeginterferon alfa-2b compared with anagrelide in patients with ET who are resistant or cannot tolerate HU. Clinical Trial Registration: NCT04285086 (ClinicalTrials.gov).

99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer.

UNLABELLED: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and small-molecule radiopharmaceuticals targeting PSMA rapidly detect the location and extent of disease. Here we evaluated preclinically 4 novel (99m)Tc-labeled small-molecule inhibitors of PSMA with the potential for clinical translation for molecular imaging of prostate cancer in humans. METHODS: Four PSMA inhibitors derived from the glutamate-urea-glutamate or glutamate-urea-lysine pharmacophores conjugated to CIM or TIM chelators were radiolabeled with (99m)Tc and evaluated in vitro and in vivo. RESULTS: High-affinity, saturable binding to PSMA on LNCaP cells was observed with Kd values of 0.64 ± 0.46 nM for (99m)Tc-MIP-1427, 1.07 ± 0.89 nM for (99m)Tc-MIP-1404, 1.75 ± 0.32 nM for (99m)Tc-MIP-1428, and 4.35 ± 0.35 nM for (99m)Tc-MIP-1405. (99m)Tc-labeled PSMA inhibitors did not bind human prostate cancer PC3 cells, which lack PSMA, demonstrating specificity, and binding was abolished with 2-(phosphonomethyl)pentanedioic acid (PMPA), a structurally unrelated PSMA inhibitor. (99m)Tc-labeled PSMA inhibitors were shown to internalize at 37 °C. Uptake in LNCaP xenografts ranged from 9.3% to 12.4% injected dose per gram at 1 h after injection and from 7.2% to 11.0% at 4 h, with tumor-to-blood ratios ranging from 29:1 to 550:1 and tumor-to-skeletal muscle ratios ranging from 31:1 to 157:1 at 4 h. (99m)Tc-MIP-1404 exhibited the best combination of high tumor uptake and rapid clearance from kidney and nontarget tissues. (99m)Tc-MIP-1404 specifically bound to PSMA in vivo as demonstrated by the absence of uptake in PC3 xenografts and by competition with PMPA. SPECT/CT imaging corroborated the tissue distribution results, demonstrating uptake only in PSMA-expressing kidney and tumor tissue and clearance through the urinary bladder. CONCLUSION: These (99m)Tc-labeled radiopharmaceuticals targeting PSMA may provide a SPECT molecular imaging option to assist in the initial diagnosis of prostate cancer and the management of patient care by monitoring disease progression.

Synthesis and SAR of 99mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates.

Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel (99m)Tc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells. IC(50) values ranged from 3.8 ± 2 to >2000 nM. A linker between the SAAC chelate and pharmacophore was required for high affinity binding. However, extending the length of the linker did not substantially improve binding. PSMA binding was also influenced by the nature of the SAAC chelate. One of the most potent compounds, 23b (IC(50)=4.8 ± 2.7 nM), was radiolabeled with technetium tricarbonyl ({(99m)Tc(CO)(3)}(+)) to afford the {(99m)Tc(CO)(3)}(+) complex in excellent yield and high purity. This effort has led to the identification of a diverse series of promising high affinity {(99m)Tc(CO)(3)}(+) radiolabeled PSMA inhibitors.

Synthesis and SAR of novel Re/99mTc-labeled benzenesulfonamide carbonic anhydrase IX inhibitors for molecular imaging of tumor hypoxia.

Carbonic anhydrase IX (CA-IX) is upregulated in cancer in response to the hypoxic tumor microenvironment, making it an attractive molecular target for the detection of hypoxic solid tumors. A series of small molecule benzenesulfonamide based CA-IX inhibitors containing novel tridentate chelates complexed with the M(CO)(3) core (M = Re or (99m)Tc) were designed and synthesized. The in vitro binding affinity of the benzenesulfonamide rhenium complexes yielded IC(50) values ranging from 3 to 116 nM in hypoxic CA-IX expressing HeLa cells. One of the most potent compounds, 3d (IC(50) = 9 nM), was radiolabeled with technetium tricarbonyl ({(99m)Tc(CO)(3)}(+)) to afford the {(99m)Tc(CO)(3)}(+) complex in excellent yield and high purity. (99m)Tc(CO)(3)-3d bound specifically to CA-IX expressing hypoxic HeLa cells. This effort led to the identification of a diverse series of promising high affinity {(99m)Tc(CO)(3)}(+) radiolabeled CA-IX inhibitors with the potential to significantly impact diagnosis, staging, and treatment selection of hypoxic solid tumors.

Novel polar single amino acid chelates for technetium-99m tricarbonyl-based radiopharmaceuticals with enhanced renal clearance: application to octreotide.

Single amino acid chelate (SAAC) systems for the incorporation of the M(CO)(3) moiety (M = Tc/Re) have been successfully incorporated into novel synthetic strategies for radiopharmaceuticals and evaluated in a variety of biological applications. However, the lipophilicity of the first generation Tc(CO)(3)-dipyridyl complexes has resulted in substantial hepatobiliary uptake when either examined as lysine derivatives or integrated into biologically active small molecules and peptides. Here we designed, synthesized, and evaluated novel SAAC systems that have been chemically modified to promote overall Tc(CO)(3)L(3) complex hydrophilicity with the intent of enhancing renal clearance. A series of lysine derived SAAC systems containing functionalized polar imidazole rings and/or carboxylic acids were synthesized via reductive alkylation of the epsilon amino group of lysine. The SAAC systems were radiolabeled with (99m)Tc, purified, and evaluated for radiochemical stability, lipophilicity, and tissue distribution in rats. The log P values of the (99m)Tc complexes were determined experimentally and ranged from -0.91 to -2.33. The resulting complexes were stable (>90%) for at least 24 h. Tissue distribution in normal rats of the lead (99m)Tc complexes demonstrated decreased liver (<1 %ID/g) and gastrointestinal clearance (<1.5%ID/g) and increased kidney clearance (>15 %ID/g) at 2 h after injection compared to the dipyridyl lysine complex (DpK). One of the new SAAC ligands, [(99m)Tc]bis-carboxymethylimidazole lysine, was conjugated to the N-terminus of Tyr-3 octreotide and evaluated for localization in nude mice bearing AR42J xenografts to examine tissue distribution, tumor uptake and retention, clearance, and route of excretion for comparison to (111)In-DOTA-Tyr-3-octreotide and (99m)Tc-DpK-Tyr-3-octreotide. (99m)Tc-bis-(carboxymethylimidazole)-lysine-Tyr-3-octreotide exhibited significantly less liver uptake and gastrointestinal clearance compared to (99m)Tc-DpK-Tyr-3-octreotide while maintaining tumor uptake in the same mouse model. These novel chelators demonstrate that lipophilicity can be controlled and organ distribution significantly altered, opening up broad application of these novel SAAC systems for radiopharmaceutical design.

Preclinical evaluation of novel glutamate-urea-lysine analogues that target prostate-specific membrane antigen as molecular imaging pharmaceuticals for prostate cancer.

Prostate-specific membrane antigen (PSMA) is expressed in normal human prostate epithelium and is highly up-regulated in prostate cancer. We previously reported a series of novel small molecule inhibitors targeting PSMA. Two compounds, MIP-1072, (S)-2-(3-((S)-1-carboxy-5-(4-iodobenzylamino)pentyl)ureido)pentanedioic acid, and MIP-1095, (S)-2-(3-((S)-1carboxy-5-(3-(4-iodophenyl)ureido)pentyl)ureido)pentanedioic acid, were selected for further evaluation. MIP-1072 and MIP-1095 potently inhibited the glutamate carboxypeptidase activity of PSMA (K(i) = 4.6 +/- 1.6 nmol/L and 0.24 +/- 0.14 nmol/L, respectively) and, when radiolabeled with (123)I, exhibited high affinity for PSMA on human prostate cancer LNCaP cells (K(d) = 3.8 +/- 1.3 nmol/L and 0.81 +/- 0.39 nmol/L, respectively). The association of [(123)I]MIP-1072 and [(123)I]MIP-1095 with PSMA was specific; there was no binding to human prostate cancer PC3 cells, which lack PSMA, and binding was abolished by coincubation with a structurally unrelated NAALADase inhibitor, 2-(phosphonomethyl)pentanedioic acid (PMPA). [(123)I]MIP-1072 and [(123)I]MIP-1095 internalized into LNCaP cells at 37 degrees C. Tissue distribution studies in mice showed 17.3 +/- 6.3% (at 1 hour) and 34.3 +/- 12.7% (at 4 hours) injected dose per gram of LNCaP xenograft tissue, for [(123)I]MIP-1072 and [(123)I]MIP-1095, respectively. [(123)I]MIP-1095 exhibited greater tumor uptake but slower washout from blood and nontarget tissues compared with [(123)I]MIP-1072. Specific binding to PSMA in vivo was shown by competition with PMPA in LNCaP xenografts, and the absence of uptake in PC3 xenografts. The uptake of [(123)I]MIP-1072 and [(123)I]MIP-1095 in tumor-bearing mice was corroborated by single-photon emission computed tomography/computed tomography (SPECT/CT) imaging. PSMA-specific radiopharmaceuticals should provide a novel molecular targeting option for the detection and staging of prostate cancer.

Comprehensive radiolabeling, stability, and tissue distribution studies of technetium-99m single amino acid chelates (SAAC).

Technetium tricarbonyl chemistry has been a subject of interest in radiopharmaceutical development over the past decade. Despite the extensive work done on developing chelates for Tc(I), a rigorous investigation of the impact of changing donor groups and labeling conditions on radiochemical yields and/or distribution has been lacking. This information is crucially important if these platforms are going to be used to develop molecular imaging probes. Previous studies on the coordination chemistry of the {M(CO)(3)}(+) core have established alkylamine, aromatic nitrogen heterocycles, and carboxylate donors as effective chelating ligands. These observations led to the design of tridentate ligands derived from the amino acid lysine. Such amino acid analogues provide a tridentate donor set for chelation to the metal and an amino acid functionality for conjugation to biomolecules. We recently developed a family of single amino acid chelates (SAAC) that serve this function and can be readily incorporated into peptides via solid-phase synthesis techniques. As part of these continuing studies, we report here on the radiolabeling with technetium-99m ((99m)Tc) and stability of a series of SAAC analogues of lysine. The complexes studied include cationic, neutral, and anionic complexes. The results of tissue distribution studies with these novel complexes in normal rats demonstrate a range of distribution in kidney, liver, and intestines.

A robust strategy for the preparation of libraries of metallopeptides. A new paradigm for the discovery of targeted molecular imaging and therapy agents.

A robust method for synthesizing structurally diverse metallopeptide libraries using a Re(i) complex of a non-natural amino acid was developed as a way to accelerate the discovery of novel molecular imaging probes.

Synthesis and evaluation of a series of 99mTc(CO)3+ lisinopril complexes for in vivo imaging of angiotensin-converting enzyme expression.

In animal models of cardiac disease and in human congestive heart failure, expression of angiotensin-converting enzyme (ACE) is upregulated in the failing heart and has been associated with disease progression leading to cardiac failure and fibrosis. To develop probes for imaging ACE expression, a series of di(2-pyridylmethyl)amine (D) chelates capable of binding M(CO)3+ (M = technetium, rhenium) was conjugated to lisinopril by acylation of the epsilon-amine of the lysine residue with a series of di(2-pyridylmethylamino)alkanoic acids where the distance of the chelator from the lisinopril core was investigated by varying the number of methylene spacer groups to produce di(2-pyridylmethyl)amine(Cx)lisinopril analogs: D(C4)lisinopril, D(C5)lisinopril, and D(C8)lisinopril. The inhibitory activity of each rhenium complex was evaluated in vitro against purified rabbit lung ACE and was shown to vary directly with the length of the methylene spacer: Re[D(C8)lisinopril], inhibitory concentration of 50% (IC50) = 3 nM; Re[D(C5)lisinopril], IC50 = 144 nM; and Re[D(C4)lisinopril], IC50 = 1,146 nM, as compared with lisinopril, IC50 = 4 nM. The in vivo specificity for ACE was determined by examining the biodistribution of the 99mTc-[D(C8)lisinopril] analog in rats with and without pretreatment with unlabeled lisinopril. Uptake in the lungs, a tissue that constitutively expresses ACE, was 15.2 percentage injected dose per gram at 10 min after injection and was dramatically reduced by pretreatment with lisinopril, supporting ACE-mediated binding in vivo. Planar anterior imaging analysis of 99mTc-[D(C8)lisinopril] corroborated these data. Thus, high-affinity 99mTc-labeled ACE inhibitor has been designed with potency similar to that of lisinopril and has been demonstrated to specifically localize to tissues that express ACE in vivo. This agent may be useful in monitoring ACE as a function of disease progression in relevant diseases such as heart failure.

3D QSAR (COMFA) of a series of potent and highly selective VLA-4 antagonists.

The integrin VLA-4 (alpha4,beta1) is involved in the migration of white blood cells to sites of inflammation, and is implicated in the pathology of a variety of diseases including asthma and multiple sclerosis. We report the structure-activity relationships of a series of VLA-4 antagonists that were based upon the integrin-binding sequence of the connecting segment peptide of fibronectin (Leu-Asp-Val), and of VCAM-1 (Ile-Asp-Ser), both natural ligands of VLA-4. We explore variation in the ligand derived peptide portion of these antagonists and also in the novel N-terminal cap, which have discovered through chemical optimization, and which confers high affinity and selectivity. Using the X-ray derived conformation of the Ile-Asp-Ser region of VCAM-1, we rationalize the structure-activity relationships of these antagonists using 3D QSAR (COMFA). The COMFA model was found to be highly predictive with a cross-validated R2CV of 0.7 and a PRESS of 0.49. The robustness of the model was confirmed by testing the influence of various parameters, including grid size, column filtering, as well as the role of orientation of the aligned molecules. Our results suggest that the VCAM-1 structure is useful in generating highly predictive models of our VLA-4 antagonists. The COMFA model coupled with the knowledge that the peptide amides are tolerant to methylation should prove useful in future peptidomimetic design studies.