Joseph E. Bugaj

Novel receptor-targeted fluorescent contrast agents for in vivo tumor imaging.

RATIONALE AND OBJECTIVES To evaluate the efficacy of a novel tumor receptor-specific small-peptide-near-infrared dye conjugate for tumor detection by optical imaging. METHODS A novel, near-infrared dye-peptide conjugate was synthesized and evaluated for tumor-targeting efficacy in a well-characterized rat tumor model (CA20948) known to express receptors for the chosen peptide. A simple continuous-wave optical imaging system, consisting of a near-infrared laser diode, a cooled CCD camera, and an interference filter, was used in this study. RESULTS Tumor retention of two non-tumor-specific dyes, indocyanine green and its derivatized analogue, bis-propanoic acid cyanine dye (cypate), was negligible. In contrast, the receptor-specific peptide-cypate conjugate (cytate) was retained in the CA20948 tumor, with an excellent tumor-tonormal-tissue ratio in the six rats examined. CONCLUSIONS Optical detection of tumors with a receptor-targeted fluorescent contrast agent has been demonstrated. This result represents a new direction in cancer diagnosis and patient management.

Novel fluorescent contrast agents for optical imaging of in vivo tumors based on a receptor-targeted dye-peptide conjugate platform

We have designed, synthesized, and evaluated the efficacy of novel dye-peptide conjugates that are receptor specific. Contrary to the traditional approach of conjugating dyes to large proteins and antibodies, we used small peptide-dye conjugates that target over-expressed receptors on tumors. Despite the fact that the peptide and the dye probe have similar molecular mass, our results demonstrate that the affinity of the peptide for its receptor and the dye fluorescence properties are both retained. The use of small peptides has several advantages over large biomolecules, including ease of synthesis of a variety of compounds for potential combinatorial screening of new targets, reproducibility of high purity compounds, diffusiveness to solid tumors, and the ability to incorporate a variety of functional groups that modify the pharmacokinetics of the peptide-dye conjugates. The efficacy of these new fluorescent optical contrast agents was evaluated in vivo in well-characterized rat tumor lines expressing somatostatin (sst(2)) and bombesin receptors. A simple continuous wave optical imaging system was employed. The resulting optical images clearly show that successful specific tumor targeting was achieved. Thus, we have demonstrated that small peptide-dye conjugates are effective as contrast agents for optical imaging of tumors.

Noninvasive fluorescence detection of hepatic and renal function.

A noninvasive in vivo fluorescence detection scheme was employed to continuously monitor exogenous dye clearance from the vasculature. Differentiation between normal and impaired physiological function in a rat model was demonstrated for both liver and kidney. A fiber optic transmitted light from source to ear; a second fiber optic positioned near the ear transmitted the fluorescent light to a detector system. Two model dye systems were employed in this initial study. Indocyanine green, known to be exclusively cleared from the blood stream by the liver, was excited in vivo with laser light at 780 nm. The fluorescence signal was detected at 830 nm. A characteristic clearance curve of normal hepatic function was obtained. After a partial hepatectomy of the liver, the clearance curve was extended in time, as would be expected from reduced hepatic function. In addition, fluorescein labeled poly-D-lysine, a small polymer predominantly cleared from the blood stream by the kidney, was excited in vivo with laser light at 488 nm. The fluorescence signal was detected at 518 nm. A characteristic clearance curve of normal renal function was obtained. After a bilateral ligation of the kidneys, the clearance curve remained elevated and constant, indicating little if any clearance. Thus, the feasibility of a new noninvasive method for physiological function assessment was established.

Toxicity and dosimetry of 177Lu‐DOTA‐Y3‐octreotate in a rat model

Radiolabeled somatostatin analogs have demonstrated effectiveness for targeted radiotherapy of somatostatin receptor‐positive tumors in both tumor‐bearing rodent models and humans. A radionuclide of interest for cancer therapy is reactor‐produced 177Lu (t1/2 = 6.64 d; β− [100%]). The high therapeutic efficacy of the somatostatin analog 177Lu‐DOTA‐Tyr3‐octreotate (DOTA‐Y3‐TATE, where DOTA is 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid) was previously demonstrated in a tumor‐bearing rat model (Erion et al., J. Nucl. Med. 1999;40:223P; de Jong et al., Int. J. Cancer, 2001; 92:628–633). In the current study, the toxicity and dosimetry of 177Lu‐DOTA‐Y3‐TATE were determined in both normal and tumor‐bearing rats. Doses of 177Lu‐DOTA‐Y3‐TATE ranging from 0 to 123 mCi/kg were administered to rats and complete blood counts (CBCs) and blood chemistries were analyzed out to 6 weeks. No overt signs of toxicity were observed with 177Lu‐DOTA‐Y3‐TATE (i.e., lethargy, weight loss, scruffy coat or diarrhea) at any of the dose levels. Blood chemistries and CBCs were normal except for the white blood cell counts, which showed a dose‐dependent decrease. The maximum tolerated dose was not reached at 123 mCi/kg. The biodistribution of 177Lu‐DOTA‐Y3‐TATE was determined in CA20948 rat pancreatic tumor‐bearing rats, and the data were used to estimate human absorbed doses to normal tissues. The dose‐limiting organ was determined to be the pancreas, followed by the adrenal glands. The absorbed dose to the rat CA20948 tumor was estimated to be 336 rad/mCi (91 mGy/MBq). These data demonstrate that 177Lu‐DOTA‐Y3‐TATE is an effective targeted radiotherapy agent at levels that show minimal toxicity in this rat model.

Stabilization of the Optical Tracer Agent Indocyanine Green Using Noncovalent Interactions

Indocyanine green is a medically useful dye that absorbs and fluoresces in the near infrared and has been sporadically employed clinically as an optical tracer agent for liver function evaluation and cardiac output measurements. The poor stability of this dye in aqueous solution, especially at the high concentrations needed for bolus injection, has been a hindrance in clinical application. However, by using carefully chosen macromolecular additives, the stability of these aqueous dye solutions may be enhanced significantly. Such noncovalent binding between dye and carrier molecules was found to preserve substantially the dye in aqueous solutions for several weeks with no apparent changes in the measured in vivo biological properties.

Radiotherapeutic efficacy of 153Sm-CMDTPA-Tyr3-octreotate in tumor-bearing rats☆

Abstract A number of radiolabeled somatostatin analogs have been evaluated in animal tumor models for radiotherapeutic efficacy. The majority of the agents tested have used either high-energy beta-emitters, such as Y-90 or Re-188, or the Auger electron-emitting radionuclide, In-111. Because a medium-energy beta-emitter might have equivalent efficacy compared to high-energy emitters, and lower toxicity to non-target tissues, we have evaluated the therapeutic potential of the beta-emitting nuclide, Sm-153, chelated to the somatostatin analog, CMDTPA-Tyr 3 -octreotate. Using an in vitro binding assay, this octreotate derivative was shown to have high affinity for the somatostatin subtype-2 receptor (IC 50 = 2.7 nM). Biodistribution studies in CA20948 tumor-bearing Lewis rats demonstrate that the Sm-153 labeled compound has high uptake and retention in tumor tissue (1.7% injected dose/g tissue, 4 hrs post injection) and has rapid overall clearance properties from non-target tissue. Radiotherapy studies were carried out using 153 Sm-CMDTPA-Tyr 3 -octreotate and CA20948 tumor bearing Lewis rats at 7 days post implant. Dose regimens consisting of single and multiple i.v. injections of 5.0 mCi/rat (185 MBq) were employed over a time span of 7 days. Suppression of tumor growth rate was observed in all treated animals compared to untreated controls. Greater inhibition of tumor growth was observed in animals that received multiple doses. These studies indicate that medium-energy beta-emitting isotopes have considerable potential for the treatment of somatostatin receptor-positive tumors.

Monitoring physiological function by detection of exogenous fluorescent contrast agents

An invasive in vivo fluorescence detection scheme was employed to continuously monitor exogenous dye clearance from the vasculature. This invasive physiological monitoring technique was successfully demonstrated in a rabbit model. A commercially available catheter with embedded fiber optics was employed to transmit the excitation light and detect the emitted fluorescence. The clearance of indocyanine green, known to be exclusively cleared from the blood stream by the liver, was determined invasively. The clearance curves determined by this novel invasive method replicated the clearance curves in the same animals employing the non- invasive method established previously. Thus, the feasibility of a new invasive method for physiological function assessment was established.

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.

EFFECTS OF INHALED GASES ON THE ULTRASOUND CONTRAST PRODUCED BY MICROSPHERES CONTAINING AIR OR PERFLUOROPROPANE IN ANESTHETIZED DOGS

RATIONALE AND OBJECTIVES Inhaled gas mixtures with increased amounts of oxygen cause air containing ultrasound contrast agents to lose efficacy faster than during the inhalation of air. The authors hypothesized that contrast materials containing relatively insoluble gases would decrease the effects of inhaled gases on the ultrasound contrast. METHODS Anesthetized dogs were ventilated with compressed air and different oxygen/nitrogen gas mixtures. Video densitometric analysis was performed on end diastolic ultrasound images of the heart after administration of Albunex (air-filled microspheres) or Optison (perfluoropropane-filled microspheres). RESULTS Increased concentrations of oxygen caused no change in the contrast intensity produced by Optison in the left ventricular chamber. In the myocardium, however, increases in oxygen caused Optison to produce significantly less enhancement of the myocardial tissue. CONCLUSIONS The use of perfluoropropane within albumin microspheres prevented the effects of inhaled gas mixtures on contrast produced within the left ventricular chamber. In the myocardium, increased concentrations of oxygen in the inhaled gas mixtures reduce contrast intensity.

New approach to optical imaging of tumors

Site specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effect of drugs, and enhances the contrast between normal and diseased tissues. In optical medicine, biocompatible dyes can be used as phototherapeutics or as contrast agents. Previous studies have shown that the use of covalent or non-covalent dye conjugates of carriers such as antibiodies, liposomes, and polysaccharides improves the delivery of such molecules to tumors. However, large biomolecules can elicit adverse immunogenic reactions and also result in long blood clearance times, delaying visualization of target tissues. A viable alternative to this strategy is to use small bioactive molecule-dye conjugates. These molecules have several advantages over large biomolecules, including ease of synthesis of a variety of high purity compounds for combinatorial screening of new targets, enhanced diffusivity to solid tumors, and the ability to affect the pharmacokinetics of the conjugates by minor structural changes. Thus, we conjugated a near infrared absorbing dye to several bioactive peptides that specifically target overexpressed tumor receptors in established rat tumor lines. High tumor uptake of the conjugates was obtained without loss of either the peptide receptor affinity or the dye fluorescence. These findings demonstrate the efficacy of a small peptide-dye conjugate strategy for in vivo tumor imaging. Site-specific delivery of photodynamic therapy agents may also benefit from this approach.