CAMEL Research Interests
Major Scientific Achievements
CAMEL has developed a new type of MRI contrast agent for molecular imaging.
These contrast agents are detected through PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST),
which is an innovative alternative to traditional MRI contrast agents. We have invented
irreversible, responsive PARACEST MRI contrast agents that detect as little as single nanomolar concentrations of an enzyme,
which overcomes the low sensitivity of MRI for molecular imaging. We can achieve this level of detection sensitivity because our
agents respond to an enzyme by undergoing an irreversible change in covalent chemical structure, typically through enzymatic cleavage of a ligand.
Multiple PARACEST agents can be selectively detected during the same MRI scan session, so that an
unresponsive "control" agent may be co-administered to a patient along with our responsive agent.
We have exploited this advantage to create PARACEST agents that accurately measure metabolites and pH of in vivo
tissues regardless of the concentration of the agent or other tissue conditions. As an analogy, multiple optical imaging agents
that emit different colors have revolutionized histopathology and cell biology. The ability to detect multiple responsive
PARACEST MRI contrast agents during a single MRI scan session has the same potential to revolutionize in vivo molecular imaging.
CAMEL has also developed rapid MRI methods that detect PARACEST agents, and we are one of the first research teams that can routinely
detect PARACEST MRI contrast agents in vivo. We have also developed new synthesis methods that incorporate a metal chelate
at any position within a peptide, which provides rapid production of contrast agents for MRI, PET, and SPECT.
Therefore, we have developed the methodologies required to apply molecular imaging to a broad range of pathologies and
therapeutic assessments. The continued development of these methodologies and applications of these methodologies to biomedical diagnoses are described below.
1. PARACEST MRI contrast agents
Chemical Exchange Saturation Transfer (CEST) is a promising alternative to relaxivity-based contrast mechanisms for Magnetic Resonance Imaging (MRI).1
CEST MRI contrast agents possess one or more hydrogen protons that exchange with water (Figure 1). Saturation of the MR frequency of
one of these protons, followed by chemical exchange, can reduce the MR signal of the water. PARACEST (PARAmagnetic CEST) agents include
a paramagnetic lanthanide ion that shifts the MR frequency of the exchangeable proton to unique values to facilitate selective detection.
2-4 Furthermore, several types of chemical functional groups with exchangeable hydrogens may be used to generate
the PARACEST effect, which expands the utility of these agents for molecular imaging. CAMEL has developed a variety of
PARACEST MRI contrast agents to address a range of biomedical applications:
Figure 1. The PARACEST mechanism
1A. PARACEST MRI contrast agents that detect enzyme activity
We have developed an enzyme-responsive PARACEST MRI contrast agent that converts an amide to an amine during the cleavage
of the agent's peptidyl ligand by the caspase-3 enzyme, which is an important biomarker of apoptosis (Figure 2).5
This irreversible change was monitored with MRI by observing the disappearance of the PARACEST effect from the amide or the
appearance of the PARACEST effect from the amine (Figure 3). An unresponsive PARACEST agent was included in the reaction to
serve as an internal control. Michaelis-Menten kinetics analysis revealed that the PARACEST agent can preferentially detect
caspase-3 relative to other members of the caspase family of enzymes, and that the catalytic efficiency of caspase-3 for this
MRI contrast agent is an order of magnitude better than the catalytic efficiency for a commercially-available fluorescent agent.6
By exploiting the rapid catalytic efficiency of caspase-3, we detected as little as 5 nM of the enzyme. This provides a new standard for
molecular imaging with PARACEST agents, which are normally limited to the detection of biomarkers that are present at millimolar concentrations.
Figure 2. Mechanism of a PARACEST MRI contrast agent that can detect caspase-3
Figure 3. A CEST Spectrum demonstrates the detection of caspase-3
through changes in the PARACEST effect of the MRI contrast agent
We have used the same methodology to detect urokinase Plasminogen Activator, which is a biomarker of metastatic breast cancer and one of the 88 known exopeptidases in the human proteome.6,7
We hae also developed a new synthesis method to facilitate the detection of selected candidates among the 630 known human endopeptidases.
These studies establish that our fundamentally new approach constitutes a platform technology that can detect a variety of protease enzymes that are biomarkers of many biomedical applications.
1B. Metabolite- and pH-responsive MRI contrast agents
We have developed a PARACEST MRI contrast agent that can detect nitric oxide.8 This agent possesses an amine and amide functional group that each
show a PARACEST effect. The orthoamino anilide moiety of this PARACEST agent reacts with the autooxidative product of nitric oxide to form a triazine group,
which removes the exchangeable hydrogens from the agent and causes a disappearance of both PARACEST effects. This loss of PARACEST contrast can be monitored
with MRI to track the production of nitric oxide.
The PARACEST effects of the amine and amide groups of this agent have opposite dependencies on pH.9 The ratio of these two PARACEST
effects can be used to measure pH within a physiologically relevant range. Measurements of physiological pH values with other MRI contrast agents
are complicated by the need to account for the concentration of the pH-responsive agent.10 Our ratiometric approach of two PARACEST effects from
the single agent does not require a separate measurement of concentration. We have successfully applied this PARACEST agent to measure the pH in a
subcutaneous tumor mouse model (Figure 4).
Figure 4. Measurement of tumor pH with PARACEST MRI
1C. PARACEST DCE MRI to assess tumor angiogenesis
Dynamic Contrast Enhanced MRI (DCE MRI) can evaluate tumor response to anti-angiogenic therapies by measuring changes in vascular permeabilities
that cause changes in the rate of uptake of MRI contrast agents in tumor tissues (Figure 5).11-14 Contrast agents with different sizes may be
used to probe different ranges of vascular permeabilities.15,16 However, standard DCE MRI methods assume that the entire tumor has a
uniform hematocrit (the fraction of blood volume occupied by red blood cells).17 Because immature and hypoxic tumor vessels often have the lowest hematocrit,18-24
the high permeabilities of these vessels are underestimated, which results in an underestimation of tumor angiogenesis. To overcome this
fundamental flaw in standard DCE MRI, we have simultaneously applied macromolecular and small-molecule PARACEST agents to mouse models of metastatic
MDA-MB-231 human breast tumors, and we have simultaneously tracked the pharmacokinetics of both agents in an interleaved
fashion during a single MRI scan session (Figure 6).25 The ratio of the DCE MRI uptake profile of each agent was used to estimate tumor
vascular permeabilities. Because both agents must experience the same hematocrit, this ratiometric approach removes the effect of
the hematocrit from the analysis. As an additional benefit, the simultaneous administration and study of two PARACEST MRI contrast agents is almost
twice as fast as the serial administration of each agent, which has reduced our total experiment time
A reduced experiment time may translate to reduced costs, and may also improve patient comfort.
Figure 5. Assessment of tumor angiogenesis with DCE MRI
Figure 6. in vivo detection of two PARACEST MRI contrast agents
1D. PARACEST MRI and "theranostics"
We are integrating responsive PARACEST MRI contrast agents with studies of chemotherapeutics to investigate the new paradigm of theranostics.
Unlike traditional molecular imaging approaches that target the molecular signaling pathways of a pathology
(e.g., targeting cell receptors), we will image biomarkers that are responsible for the molecular mechanism of the pathology (e.g., detecting enzyme activity).
This provides a more robust approach to theranostics, because pathologies can often possess multiple signaling pathways but have more
limited molecular mechanisms. Examples include:
Selecting chemotherapy based on the detection of a "molecular signature":
Over 83% of metastatic breast cancers express two proteases, cathepsin-B and urokinase plasminogen activator (uPA), while less than 51%
of metastatic breast cancers express only one of these proteases.26 We are diagnosing this more specific "molecular signature" by simultaneously detecting both protease activities
within an in vivo mouse model of MDA-mb-231 breast cancer using PARACEST MRI.
We are also including a protease-unresponsive PARACEST contrast agent in a dendritic nanocarrier, along with both responsive PARACEST agents,
to track the in vivo pharmacokineics of the responsive agents. This approach may be used to select more aggressive chemotherapies and therapeutic regimens that are appropriate for agressive metastatic breast
Predicting the effect of a pH-dependent chemotherapeutic: The therapeutic efficacy of doxorubicin has been shown to depend on the pH of the tumor tissue.27
Furthermore, the dose of doxorubicin should be minimized to avoid cardiotoxicity.
We are using our PARACEST MRI method that can measure tumor pH to predict the therapeutic efficacy of
doxorubicin on eventual size regression and metastasis of breast tumors, before doxirubicin therapy is started.
This approach may spare patients from undergoing this therapeutic regimen if their breast tumors do not have the appropriate pH.
This approach may also be used to decrease the dose of doxoruicin for patients with breast tumors that have an appropriate pH.
Monitoring drug delivery:
Many new types of nanoscale drug carriers are currently under research development.
As shown in Figure 6, our technology has been used to track the delivery of two nanocarriers in the same tumor and
at the same time. By simultaneously monitoring the delivery of multiple types of drug nanocarriers to tumor tissues, the nanocarriers
can be directly compared in a single diagnosic experiment. This may provide the ability to select the best nanocarrier
for each patient.
Early detection of the response of an anti-angiogenic chemotherapeutic: A macromolecular contrast agent is sensitive to changes in vascular permeabilities
that are caused by an anti-angiogenic chemotherapy, while a small-molecule contrast agent is less sensitive to these therapeutic effects.
The ratio of vascular permeabilities measured with two PARACEST agents that are simultaneously administered and detected using DCE MRI can
remove the effect of the hematocrit, and can serve as a more specific indicator of anti-angiogenic therapeutic effects. We are applying PARACEST DCE MRI methods to a
mouse model of metastatic MDA-MB-231 with and without an anti-angiogenic chemotherapy.
We are comparing our ratiometric DC-PARACEST MRI results with results from each agent to evaluate the improvement in specificity for evaluating the early response
of the anti-angiogenic chemotherapy with PARACEST DCE MRI. This approach may accelerate the early detection of response to therapy,
so that the therapy can be continued in responding patients and discontinued in non-responding patients.
|2. Synthesis Methods for Molecular Imaging
To support our collaborators, we have synthesized and characterized many metal-labeled peptides, proteins, and nanoparticles that deliver gene therapies and anti-cancer therapeutics.
We have developed and optimized several customized synthesis methods to produce molecular imaging contrast agents for these collaborations and for CAMEL research.
2A. Peptide-DOTA syntheses
Standard synthesis methodologies can conjugate DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) to the amines of
peptides, including the N-terminus, lysine amino acids, or other amine-derivatized amino acids.29,30 However, these synthesis
methodologies can limit the diversity of peptidyl-DOTA molecular imaging agents, and are not applicable to the design of our enzyme-responsive PARACEST MRI contrast agents.
To address this limitation, we have developed a new synthesis methodology to conjugate the carboxylates of peptides to DOTA (Figure 7).31,32,33
Our methodology can be applied to solution-phase syntheses to conjugate a peptide to DOTA, or can be applied to standard solid-phase peptide synthesis (SPPS)
to "grow" a peptide onto DOTA. Our methodology can result in one or more DOTA moieties that are coupled to either end of the peptide or at any position within the
peptide's amino acid sequence. We have recently extended our methodology to incorporate DOTA as a side chain within the peptide. These synthesis methods have accelerated
our production of peptide-based molecular imaging agents for MRI, PET, and SPECT imaging.
Figure 7. Synthesis of peptide-DOTA contrast agents
2B. Conjugating DOTA to nanocarriers
MRI is an inherently insensitive imaging modality, and applications with metal-DOTA contrast agents typically require 0.1-10 mM concentrations of metal-DOTA chelates.3,34
The sensitivity of MRI contrast agents can be improved by conjugating multiple metal-DOTA chelates to a nanocarrier in order to deliver a high density
of metal ions without inducing toxicity within in vitro samples or in vivo subjects. We have exploited a new bioorthogonal chemistry approach to conjugate metal-DOTA
derivatives to a biocompatible polymer nanocarrier without the need to protect other chemical functionalities of the DOTA derivative.35
We are using this bioorthogonal synthesis method to convert our PARACEST agents from monomeric to polymeric forms, in order to boost the
sensitivity of the PARACEST effect. This method can also be used to conjugate multiple DOTA derivatives to a polymer, to detect multiple proteases, metabolites,
and/or pH with a single agent while also tracking the pharmacokinetics of the nanocarrier. This method can also be used to generate multi-modality
molecular imaging agents, by conjugating lanthanide-DOTA chelates for MRI and by conjugating 64Cu-DOTA, 99Tm-DOTA or 111In-DOTA chelates for nuclear imaging.
PAMAM dendrimers can be used to conveniently conjugate multiple lanthanide-DOTA contrast agents and other targeting moieties to the dendrimer surface.36-38
We have optimized several conjugation strategies in our laboratory to facilitate the synthesis of multifunctional and multimodality dendritic molecular imaging
agents and to create non-toxic derivatives of PAMAM dendrimers (Figure 8). For example, we have conjugated PARACEST agents to G5-PAMAM dendrimers to improve detection sensitivity
from ~5 mM for a monomeric agent to ~0.1 mM for our dendritic agent, which has greatly facilitated our in vivo PARACEST MRI studies.
Figure 8. Synthesis of a dendrimer-based MRI contrast agent
2C. High Throughput Screening
As a loner-term goal, we are also developing methods for high throughput screening of libraries of peptide-based contrast agents to identify the best
agents for biomedical imaging applications. These methods will be derived from past accomplishments in the biotechnology industry,
during which the MRI protocols were optimized to screen 15,552 samples within six hours, or over 600,000 samples within three months.39
Although high throughput screening is routinely applied to develop pharmaceuticals with great impact, the same engineering design criteria that select the best pharmaceuticals do not necessarily apply to the selection of the best diagnostic molecular imaging agents.40,41 Examples include:
Identification of molecular imaging agents with appropriate catalytic activity for an enzyme biomarker:
Pharmaceuticals are often designed to saturate the molecular target in order to cause complete inhibition of the signaling pathway or molecular mechanism.
Molecular imaging agents are often designed to avoid saturation so that the physiology is not perturbed.
Therefore, MRI contrast agents must be identified that have sufficiently rapid catalytic activity to detect specific enzyme biomarkers
and with sufficiently slow catalytic activity to avoid saturation of the enzyme.
Identification of molecular imaging agents with specific cell internalization:
Pharmaceuticals are often hydrophobic or lipophlic, which facilitates cell internalization. Molecular imaging agents are often charged,
which inhibits cell internalization. We will develop and apply high throughput screening methods to identify MRI
contrast agents with cell-targeting and cell-penetrating peptides that can facilitate the internalization into specific types of cells.
2D. Synthesis of contrast agents for other molecular imaging modalities
As a long-term goal, we are also developing new contrast agents for biomedical applications with other molecular imaging modalities. In particular, we are interested in developing
responsive optical contrast agents for biomedical investigations with new collaborators in the Biomedical Engineering Interdisciplinary Program, the Research Radiology Institute, and the
College of Optical Sciences at the University of Arizona. We also plan to employ our synthesis methods that rapidly create DOTA chelates of 64Cu,
99Tm, and 111In radionuclides for PET and SPECT imaging applications with new collaborators in the Center for Gamma-ray Imaging.
- Ward KM, Aletras AH, Balaban RS. A new class of contrast agents for MRI based on proton chemical exchange dependent saturation transfer (CEST). J Magn Reson, 2000; 143(1):79-87.
- Zhang S, Winter P, Wu K, Sherry AD. A novel europium(III)-based MRI contrast agent. J. Am. Chem. Soc., 2001;1 23(7):1517-1518.
- Zhang S, Merritt M, Woessner DE, Lenkinski RE, Sherry AD. PARACEST Agents: Modulating MRI Contrast via Water Proton Exchange. Acc. Chem Res., 2003; 36:783-790.
- Aime S, Barge A, Delli Castelli D, Fedeli F, Mortillaro A, Nielsen FU, Terreno E. Paramagnetic lanthanide(III) complexes as pH-sensitive chemical exchange saturation transfer (CEST) contrast agents for MRI applications. Magn. Reson. Med., 2002; 47(4):639-648.
- Yoo B, Pagel MD. A PARACEST MRI contrast agent to detect enzyme activity. J. Am. Chem. Soc., 2006; 128(43):14032-14033.
- Yoo B, Raam M, Rosenblum R, Pagel MD. Enzyme-responsive PARACEST MRI contrast agents: A new biomedical imaging approach for studies of the proteasome. Contrast Media Molec. Imag., 2007, 2:189-198.
- Yoo B, Sheth V, Pagel MD. An amine-derivatized, DOTA-loaded polymeric support for Fmoc Solid Phase Peptide Synthesis. Tet Lett, 2009, 50:4459-4462.
- Liu G, Lu Y, Pagel MD. Design and characterization of new irreversible responsive PARACEST MRI contrast agent that detects nitric oxide. Magn. Reson. Med., 2007; 58:1249-1256.
- Liu G, Li Y, Pagel MD. Measurement of Extracelluar pH Using A Single MRI Contrast Agent Based On PARACEST Effects. Submitted to Andrew. Chemie. Int. Ed.
- Garcia-Martin ML, Martinez GV, Raghunand N, Sherry AD, Zhang S, Gillies RJ. High resolution pHe imaging of rat glioma using pH-dependent relaxivity. MRM, 2006, 55:309-315. An application of Gd-DOTP.
- Brasch RC, Li KCP, Husband JE, Keogan MT, Neeman M, Padhani AR, Shames D, Turetschek K. In vivo Monitoring of Tumor Angiogenesis with MR Imaging, Acad. Radiol., 2000; 7:812-823.
- Taylor JS, Tofts PS, Phil D, Ruediger P, Evelhoch JL, Knopp M, Reddick WE, Runge, VM Mayr N, MR Imaging of Tumor Microcirculation: Promise for the New Millenium, JMRI 10:903-907, 1999.
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- Yuan F, Dellin M, Fukumura D. Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res, 1995, 55:3752-3756.
- Daldrup H, Shames DM, Wendland M, Okuhata Y, Link TM, Rosenau W, Lu Y, Brasch RC, Correlation of Dynamic Contrast-Enhanced Magnetic Resonance Imaging with Histologic Tumor Grade: Comparison of Macromolecular and Small-Molecular Contrast Media, Pediatr Radiol 28:67-78, 1998.
- Evelhoch JL, Key Factors in the Acquisition of Contrast Kinetic Data for Oncology, JMRI 10:254-259, 1999.
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- Gillies RJ, Gatenby RA. Hypoxia and adaptive landscapes in the evolution of carcinogenesis. Cancer Metastasis Rev, 2007, 26(2):311-317.
- Ali MM, Yoo B, Pagel MD. Tracking the relative in vivo pharmacokinetics of nanoparticles with PARACEST MRI. Molec Pharmaceutics, 2009, available on-line, hardcopy in press.
- Koblinski JE, Ahram M, Sloane BL. Unraveling the role of proteases in cancer. Clinica Chimica Acta 2000; 291:113-35.
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- Ali MM, Liu G, Shah T, Flask CA, Pagel MD. Using Two Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agents for Molecular Imaging Studies. Acc Chem Res, 2009, 42(7):915-924.
- De Leon-Rodriguez, L. M., Kovacs, Z., Dieckmann, G. R., and Sherry, A. D. (2004) Solid-phase synthesis of DOTA-peptides. Chem. Eur. J. 10, 1149-1155.
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- Yoo B, Pagel MD. A facile synthesis of alpha-amino-DOTA as a versatile molecular imaging probe. Tetrahedron Lett., 2006; 47:7327-7330.
- Yoo B, Pagel MD. Peptidyl Molecular Imaging Contrast Agents Using a New Solid Phase Peptide Synthesis Approach. Bioconj. Chem., in press.
- Yoo B, Sheth V, Pagel MD. An amine-derivatized, DOTA-loaded polymeric support for Fmoc Solid Phase Peptide Synthesis. Tet Lett, 2009, 50:4459-4462.
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- Wang SJ, Brechbiel M, Wiener EC. Characteristics of a New MRI Contrast Agent Prepared from PolyPropyleneImine Dendrimers, Generation 2. Invest. Radiol., 2003; 38:662-668.
- Venditto VJ, Regino CA, Brechbiel MW. PAMAM dendrimer based macromolecules as improved contrast agents. Mol. Pharm., 2005; 2:302-11.
- Kobayashi H, Kawamoto S, Jo SK, Bryant HL, Brechbiel MW, Star RA. Macromolecular MRI Contrast Agents with Small Dendrimers: Pharmacokinetic Differences between Sizes and Cores. Bioconjugate Chem., 2003; 14:388-394.
- Kotyk JJ, Pagel MD, Deppermann KL, Colletti RF, Hoffman NG, Yannakakis EJ, Das PK, Ackerman JJH. High-Throughput Determination of Oil Content in Corn Kernels Using Nuclear Magnetic Resonance Imaging. J. Am. Oil Chem. Soc., 2005; 82:855-862.
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