Evaluation of Developmental and Reproductive Toxicity in Rabbits for MB-102, a Fluorescent Tracer Agent Designed for Real-Time Measurement of Glomerular Filtration Rate.

The fluorescent tracer, MB-102, has been designed for the direct, real-time measurement of glomerular filtration rate. Previous studies, both in vitro and in vivo (rats, rabbits and dogs), were conducted to assess potential toxicity including single dose toxicity, mutation assay, chromosomal aberration assay, phototoxicity, local tolerance study, micronuclease assay, hERG channel changes, CNS and cardiovascular safety. The results of these studies led to a safety/toxicology profile for this agent deemed sufficient by the FDA to conduct Phase I and Phase II human clinical studies. In this paper we report on maternal toxicity and the potential effects on embryo-fetal development and the toxicokinetics of MB-102 administered daily via intravenous (bolus) injection into pregnant rabbits during the period of organogenesis gestation day 7-19. Assessment of toxicity was based on mortality, clinical observations, body weight, food consumption, reproductive performance and necropsy and cesarean section findings. Blood samples were collected for toxicokinetic evaluation. No test article findings were noted in any of these studies. The only clinical findings observed were the discoloration of skin, eyes or pelage in the 2 higher dose groups, which were considered related to the color and fluorescent properties of MB-102 and were deemed non-adverse. Exposure, as assessed by Cmax and AUC(0-6) increased in a dose dependent manner from 4.5 to 113 mg/kg/day. No accumulation of the test article was noted after multiple doses were administered. Thus, intravenous administration of MB-102 was not associated with any adverse developmental or reproductive toxicities in pregnant rabbits.

Absence of developmental or reproductive toxicity in rats for MB-102, a fluorescent tracer agent for point-of-care measurement of glomerular filtration rate.

The fluorescent tracer agent MB-102 was designed for the direct, real-time measurement of glomerular filtration rate. Previous studies, both in vitro and in vivo (rats, rabbits and dogs) have assessed single dose toxicity, phototoxicity, local tolerance, hERG channel changes, mutation, chromosomal aberration, micronuclease assays, and CNS and cardiovascular safety. The resulting safety/toxicology profile allowed FDA clearance to conduct Phase I and II human clinical studies. Herein we report on maternal toxicity and potential effects on embryo-fetal development and toxicokinetics of MB-102 administered daily via intravenous (bolus) injection into pregnant rats during organogenesis gestation day 6-17. Mortality, clinical observations, body weight, food consumption, reproductive performance, necropsy, and cesarean section findings were assessed. Blood samples were evaluated toxicokinetically. No significant findings were noted in any endpoints. The only clinical findings were skin, eye or pelage discoloration in the two higher dose groups, which were considered related to the color and fluorescent properties of MB-102 and deemed non-adverse. Exposure, assessed by Cmax and AUC(0-6), increased in a dose-dependent manner from 9 to 225 mg/kg/day. Thus, intravenous administration of MB-102 was not associated with any adverse developmental or reproductive toxicities in pregnant rats.

Next tier in vitro and in vivo nonclinical studies further elucidating the safety and toxicity profile of MB-102, a novel fluorescent tracer agent for measurement of glomerular filtration rate.

MB-102 was designed for measurement of real-time glomerular filtration rate (GFR). Previously reported in vitro and in vivo nonclinical studies clearly demonstrated negligible toxicity, resulting in FDA clearance for First-in Human, proof of concept clinical studies. The next tier of safety and toxicity studies are reported herein. MB-102 did not demonstrate any phototoxic potential in a BALB/c 3T3 mouse fibroblast study. Co-administration of MB-102 and iohexol resulted in pharmacokinetic parameters virtually identical to the values observed upon individual administration in beagle dogs. A single dose of MB-102 administered either intravenously (18.6 mg/mL) or perivenously (0.25 mL) was well-tolerated in NZ white rabbits, with no adverse inflammation or irritation. MB-102 did not induce micronuclei in polychromatic erythrocytes for rat bone marrow cells treated up to 450 mg/kg/day, the maximum feasible dose. Two separate optical imaging studies demonstrated that MB-102 distributes rapidly and thoroughly throughout the test subjects, followed by rapid clearance from the body without any preferential localization in any particular tissue or organ, with the exception of the bladder, which is totally consistent with a known GFR agent. In addition, two-week repeat intravenous (once-daily) toxicity and toxicokinetic studies were conducted in rats and beagles, with no MB-102- related effects. Thus, for the studies reported herein, there were no toxicological effects of concern for MB-102.

Pre-clinical toxicity evaluation of MB-102, a novel fluorescent tracer agent for real-time measurement of glomerular filtration rate.

The fluorescent tracer agent 3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl}pyrazine, designated MB-102, has been developed with properties and attributes for use as a direct measure of glomerular filtration rate (GFR). In comparison to known standard exogenous GFR agents in animal models, MB-102 has demonstrated an excellent correlation. A battery of toxicity tests has been completed on this new fluorescent tracer agent, including single dose toxicity studies in rats and dogs to determine overall toxicity and toxicokinetics of the compound. Blood compatibility, mutation assay, chromosomal aberration assay, and several other assays were also completed. Toxicity assessments were based on mortality, clinical signs, body weight, food consumption and anatomical pathology. Doses of up to 200-300 times the estimated human dose were administered. No test-article related effects were noted on body weight, food consumption, ophthalmic observations and no abnormal pathology was seen in either macroscopic or microscopic evaluations of any organs or tissues. All animals survived to scheduled sacrifice. Transient discoloration of skin and urine was noted at the higher dose levels in both species as expected from a highly fluorescent compound and was not considered pathological. Thus initial toxicology studies of this new fluorescent tracer agent MB-102 have resulted in negligible demonstrable pathological test article concerns.

18FDG-PET predicts pharmacodynamic response to OSI-906, a dual IGF-1R/IR inhibitor, in preclinical mouse models of lung cancer.

PURPOSE: To evaluate 2-deoxy-2-[(18)F]fluoro-d-glucose positron emission tomography imaging ((18)FDG-PET) as a predictive, noninvasive, pharmacodynamic (PD) biomarker of response following administration of a small-molecule insulin-like growth factor-1 receptor and insulin receptor (IGF-1R/IR) inhibitor, OSI-906. EXPERIMENTAL DESIGN: In vitro uptake studies of (3)H-2-deoxy glucose following OSI-906 exposure were conducted evaluating correlation of dose with inhibition of IGF-1R/IR as well as markers of downstream pathways and glucose metabolism. Similarly, in vivo PD effects were evaluated in human tumor cell line xenografts propagated in athymic nude mice by (18)FDG-PET at 2, 4, and 24 hours following a single treatment of OSI-906 for the correlation of inhibition of receptor targets and downstream markers. RESULTS: Uptake of (3)H-2-deoxy glucose and (18)FDG was significantly diminished following OSI-906 exposure in sensitive tumor cells and subcutaneous xenografts (NCI-H292) but not in an insensitive model lacking IGF-1R expression (NCI-H441). Diminished PD (18)FDG-PET, collected immediately following the initial treatment agreed with inhibition of pIGF-1R/pIR, reduced PI3K (phosphoinositide 3-kinase) and MAPK (mitogen activated protein kinase) pathway activity, and predicted tumor growth arrest as measured by high-resolution ultrasound imaging. CONCLUSION: (18)FDG-PET seems to serve as a rapid, noninvasive PD marker of IGF-1R/IR inhibition following a single dose of OSI-906 and should be explored clinically as a predictive clinical biomarker in patients undergoing IGF-1R/IR-directed cancer therapy.

Novel approach in radionuclide tumor therapy: dose enhancement by high Z-element contrast agents.

PURPOSE: The aim of this study was to first calculate the dose-enhancement effect from internalized radiation by the presence of exogenous contrast media using Monte Carlo simulations, and then provide in vitro proof-of-concept for this novel method of radiation-dose enhancement. MATERIALS AND METHODS: The Monte Carlo program EGSnrc (Electron Gamma Shower) was used to simulate the interaction of internalizing radiation with iodine (I) or gadolinium (Gd) containing contrast media. Experimentally, the doseenhancement effect induced by I or Gd was evaluated in cell culture assays using internalizing peptides chelated with beta- emitting radionuclides and clinically available contrast media. RESULTS: Monte Carlo simulations predicted significant enhancement (approximately 70-340%) of radiation dose in the presence of high Zelement contrast media. This enhancement is radiation and Z-element dependent. Calculations showed that in the presence of contrast media, low-energy radionuclides favor localization of secondary particles, whereas higher energy beta- emitters localize radiation by reducing the pathway of the primary beta-particle. The dose enhancement was verified in vitro in two cell lines. CONCLUSIONS: Monte Carlo simulations in parallel with in vitro studies provide proof-of-principle for dose enhancement that occurs when utilizing an internalized source of radiation followed by the addition of exogenous contrast media. This dose enhancement is both radiation and Z-element dependent.

Novel bioactive and stable neurotensin peptide analogues capable of delivering radiopharmaceuticals and molecular beacons to tumors.

The prevalence of neurotensin receptor (NTR) in several human tumors makes it an attractive target for the delivery of cytotoxic drugs and imaging agents. Native neurotensin (NT) is a tridecapeptide that binds to NTR and induces tumor growth. Unfortunately, NT has a short plasma half-life, which hinders its use for in vivo biomedical applications. Numerous reports suggest that Arg(8)-Arg(9) and Tyr(11)-Ile(12) amide bonds are particularly susceptible to degradation by proteolytic enzymes. Predicated on this observation, we substituted Arg(8), Arg(9), and Ile(12) amino acids with the corresponding commercially available mimics. These surrogate amino acids are amenable to standard Fmoc peptide synthesis strategy, and the resulting compounds are stable in biological media for >4 h and bind to NTR with high affinity. Furthermore, conjugating DTPA to the new peptides and subsequent labeling with (111)In-DTPA for nuclear imaging or fluorescein for optical imaging did not diminish the NTR binding affinities of the peptides. In vivo biodistribution of a representative (111)In-DTPA-NT peptide analogue in SCID mice bearing NTR-positive human adenocarcinoma (HT29) xenograft shows that the compound was primarily retained in tumor tissue (2.2% ID/g) and the kidneys (4.8% ID/g) at 4 h postinjection. Coinjection of cold NT and the radiolabeled NT peptide analogue inhibited the tumor but not the kidney uptake, demonstrating that retention of the radiolabeled compound in tumor tissue was mediated by NTR specific uptake while it accumulates in the kidneys by a nonspecific mechanism. These findings show that the new NT peptide analogues are robust and can deliver imaging agents to NTR-positive tumors such as pancreatic cancer.

Preclinical comparison of (111)In-labeled DTPA- or DOTA-bombesin analogs for receptor-targeted scintigraphy and radionuclide therapy.

UNLABELLED: The 14-amino-acid peptide bombesin (BN) has a high affinity for the gastrin-releasing peptide (GRP) receptor that is expressed by a variety of tumors. Recently, high densities of GRP receptors were identified by in vitro receptor autoradiography in human prostate and breast carcinomas using [(125)I-Tyr(4)]BN as radioligand. Radiometal-labeled diethylenetriaminepentaacetic acid (DTPA)-BN derivatives are potentially useful radioligands for receptor-targeted scintigraphy and radiotherapy of GRP receptor-expressing tumors. METHODS: [DTPA-Pro(1),Tyr(4)]BN (A), [DOTA-Pro(1),Tyr(4)]BN (B), [DTPA-epsilon-Lys(3),Tyr(4)]BN (C), and [DOTA-epsilon-Lys(3),Tyr(4)]BN (D) (where DOTA is dodecanetetraacetic acid) were synthesized and studied for competition with binding of [(125)I-Tyr(4)]BN to the GRP receptor. The (111)In-labeled BN analogs were studied in vitro for binding and internalization by GRP receptor-expressing CA20948 and AR42J pancreatic tumor cells as well as in vivo for tissue distribution in rats. Specific tissue binding was tested by coinjection of 0.1 mg [Tyr(4)]BN. RESULTS: All BN analogs competitively inhibited the binding of [(125)I-Tyr(4)]BN to the GRP receptor with 50% inhibitory concentration values in the range of 2-9 nmol/L. All (111)In-labeled analogs showed high and specific time- and temperature-dependent binding and internalization by CA20948 and AR42J cells. In in vivo studies, high and specific binding was found in GRP receptor-positive tissues such as pancreas (0.90, 1.2, 0.54, and 0.79 percentage injected dose per gram for A-D, respectively). In a rat model, the AR42J tumor could clearly be visualized by scintigraphy using [(111)In-DTPA-Pro(1),Tyr(4)]BN as the radioligand. Although [(111)In-DOTA-Pro(1),Tyr(4)]BN showed the highest uptake of radioactivity in GRP receptor-positive tissues as well as higher target-to-blood ratios, [(111)In-DTPA-Pro(1),Tyr(4)]BN was easier to handle and is more practical to use. Therefore, we decided to start phase I studies with this DTPA-conjugated radioligand. CONCLUSION: [(111)In-DTPA-Pro(1),Tyr(4)]BN is a promising radioligand for scintigraphy of GRP receptor-expressing tumors. We are currently performing a phase I study on patients with invasive prostate carcinoma.

DOTA-D-Tyr(1)-octreotate: a somatostatin analogue for labeling with metal and halogen radionuclides for cancer imaging and therapy.

The goal of this study was to evaluate a somatostatin receptor ligand, DOTA-D-Tyr(1)-octreotate (DOTA-DY1-TATE), that has the chelator 1,4,7,10-tetraazacyclotetradecane-N,N',N'',N'"-tetraacetic acid (DOTA) attached to the D-Tyr(1) residue, allowing radiolabeling with both radiohalogens and radiometals. A potential advantage of having a chelator attached to the Tyr(1) residue is that halogen radiolabels may residualize or remain trapped in tumor cells rather than clear from the tumor. DOTA-DY1-TATE was synthesized by solid-phase methods and radiolabeled with (61)Cu, (64)Cu, and (125)I in high radiochemical purity and specific activity. A competitive binding assay demonstrated that (nat)Cu-DOTA-DY1-TATE and DOTA-(nat)I-DY1-TATE had comparable affinity to (nat)In-DTPA-OC in AR42J rat pancreatic tumor cells membranes. (61)Cu-DOTA-DY1-TATE had a dissociation constant (K(d)) of 176.4 pM and a receptor concentration (B(max)) of 244.4 fmol/mg. A tumor uptake of 1.515 %ID/g was determined for (64)Cu-DOTA-DY1-TATE and 0.814 %ID/g for DOTA-(125)I-DY1-TATE in AR42J tumor bearing Lewis rats at 1 h postinjection. DOTA-(125)I-DY1-TATE remained in the tumor at a higher concentration out to 4 h postinjection, suggesting that the iodine may have residualized in the tumor cells. MicroPET imaging of (64)Cu-DOTA-DY1-TATE in AR42J tumor bearing rats and SCID mice at 2 h postinjection showed significant uptake and good contrast in the thigh tumors in the rat model and in the neck and thigh tumors of the mouse. This study demonstrates that DOTA-DY1-TATE is a somatostatin analogue that can be labeled with both metal and halogen radionuclides, and its (64)Cu- and (125)I-radiolabeled compounds showed somatostatin receptor-mediated uptake in normal and tumor tissues.

Synthesis, in vitro receptor binding, and in vivo evaluation of fluorescein and carbocyanine peptide-based optical contrast agents.

Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effects of drugs and enhances the contrast between normal and pathologic tissues. One approach to achieve selectivity is to target overexpressed receptors on the membranes of tumor cells and to visualize the tumors by a noninvasive optical imaging method. Accordingly, we conjugated fluorescein and carbocyanine dyes to somatostatin and bombesin receptor-avid peptides and examined their receptor binding affinities. We also prepared potential dual imaging probes consisting of a bioactive peptide for tumor targeting, a biocompatible dye for optical imaging, and a radioactive or paramagnetic metal chelator for scintigraphic or magnetic resonance imaging of tumors. Using these approaches, the resulting carbocyanine derivatives of somatostatin and bombesin analogues retained high binding for their respective receptors. Further evaluation of representative molecules in rats bearing somatostatin- and bombesin-positive tumors showed selective uptake of the agents by the tumor cells. Unlike carbocyanine derivatives, the receptor binding of fluorescein-somatostatin peptide conjugates was highly sensitive to the type of linker and the site of fluorescein attachment on the nonreceptor binding region of the peptide. In general, the presence of flexible linkers disrupted binding affinity, possibly due to the interaction of the linker's thiourea group with the peptide's cyclic disulfide bond. While the receptor binding affinity of the dual probes was not dependent on the type of chelating group examined, it was affected by the relative positions of fluorescein and chelator on the lysine linker. For somatostatin compounds, best results were obtained when the chelator was on the alpha-amino lysine linker and fluorescein was on the epsilon-amino group. In contrast, conjugation of the chelator to epsilon- and fluorescein to the alpha-amino lysine linker of bombesin peptides resulted in high receptor binding. These findings indicate that despite their small size, conjugation of dyes to truncated somatostatin and bombesin peptide analogues results in promising diagnostic agents that retain high receptor binding activity in vitro. The results further show that these contrast agents can selectively and specifically localize in receptor-positive tumors in rat models.