Balancing the intermolecular forces in peptide amphiphiles for controlling self-assembly transitions.

While the influence of alkyl chain length and headgroup size on self-assembly behaviour has been well-established for simple surfactants, the rational control over the pH- and concentration-dependent self-assembly behaviour in stimuli responsive peptides remains an elusive goal. Here, we show that different amphiphilic peptides can have similar self-assembly phase diagrams, providing the relative strengths of the attractive and repulsive forces are balanced. Using palmitoyl-YYAAEEEEK(DO3A:Gd)-NH2 and palmitoyl-YAAEEEEK(DO3A:Gd)-NH2 as controls, we show that reducing hydrophobic attractive forces through fewer methylene groups in the alkyl chain will lead to a similar self-assembly phase diagram as increasing the electrostatic repulsive forces via the addition of a glutamic acid residue. These changes allow creation of self-assembled MRI vehicles with slightly different micelle and nanofiber diameters but with minimal changes in the spin-lattice T1 relaxivity. These findings reveal a powerful strategy to design self-assembled vehicles with different sizes but with similar self-assembly profiles.

Designer peptides: learning from nature.

Recent advances in designing peptide ligands for therapeutic targets are making peptides an attractive alternative to small molecules and proteins. It is now common to see peptides developed with affinities comparable to antibodies and specificities much better than small molecules or antibodies. This is especially true in the case of tumor targeting cytotoxic drugs or targeted diagnostics where peptides can be used as a delivery vehicle for drugs or diagnostics. Moreover, lessons learned from nature in understanding peptide ligands are proving to be useful in designing better antibodies and small molecule therapeutics.

A distinct strategy to generate high-affinity peptide binders to receptor tyrosine kinases.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Synthesis, characterization, and imaging performance of a new class of macrocyclic hepatobiliary MR contrast agents.

RATIONALE AND OBJECTIVES: To investigate the effect of substituent lipophilicity, substituent position, and overall charge on the hepatobiliary clearance and tolerance of a series of aromatic ring-containing macrocyclic Gd chelates to select a candidate compound for evaluation as a hepatobiliary imaging agent. METHODS: Hepatobiliary clearance was studied in rats. Tissue distribution and tolerance were studied in mice. Imaging was performed in cats, rabbits, and Rhesus monkeys using T1-weighted pulse sequences or T1-weighted breath-hold pulse sequences. RESULTS: All the compounds were excreted bimodally. Gd-2,5-BPA-DO3A (15d) was found to have the optimal combination of hepatobiliary clearance (47% in rats, 29% in mice) and tolerance (minimum lethal dose 5.0 mmol/kg). Initial imaging studies in cats demonstrated the feasibility of Gd-2,5-BPA-DO3A for hepatic imaging. In rabbits with implanted VX-2 adenocarcinoma as a model for metastatic liver disease, Gd-2,5-BPA-DO3A provided sustained hepatic signal intensity (SI) enhancement and lesion conspicuity over a 120-minute imaging time course. In Rhesus monkeys with normal liver function, Gd-2,5-BPA-DO3A afforded sustained hepatic SI enhancement and a time-dependent increase in gallbladder SI over the entire 90-minute imaging time course. CONCLUSIONS: Gd-2,5-BPA-DO3A provides dramatic and sustained SI enhancement of hepatic tissue in cats, rabbits, and Rhesus monkeys that was superior in all respects to the extracellular space MRI agent, Gd-HP-DO3A, that was employed as a control.

Caenorhabditis elegans as an alternative animal species.

Caenorhabditis elegans has proven useful in toxicity testing of known toxicants, but its potential for assessing the toxicity of new pharmaceuticals is relatively unexplored. In this study the procedures used in aquatic testing of toxicants were modified to permit testing of small amounts (<40 mg) of gadolinium-based magnetic resonance imaging (MRI) compounds. Five blinded compounds were tested. The toxicity of these compounds determined using C. elegans was compared to existing mammalian test system data (minimum lethal dose [MLD] values for mice). Four of five compounds tested had the same relative sensitivity with C. elegans as with the mouse test system. Testing with C. elegans is efficient and could markedly reduce the cost of screening potentially useful compounds.

Utilization of the nephrectomized mouse for determining threshold effects of MRI contrast agents.

The tissue concentration of an extravascularly distributed MRI contrast agent required to achieve a 20% change in the MRI signal intensity (SI) of skeletal muscle was determined using radiolabeled gadoteridol administered to nephrectomized mice. This minimal change in the quantified SI was reliably detected qualitatively in the MR muscle images. MR images of muscle were acquired following each intravenous injection of six sequential doses of 0.8 micromol of 153Gd-labeled gadoteridol. A 2.0 T imaging spectrometer and a T1-weighted spin-echo pulse sequence were used to acquire the MR images. After imaging, the injected 153Gd in muscle was measured, and the 153Gd assay results were used to determine the gadoteridol concentration in muscle following each injection. The muscle concentrations of gadoteridol were then correlated to the quantified enhanced MR SI of muscle. Using the 20% factor, it was concluded that the amount of gadoteridol necessary to achieve a reliable change in the SI of muscle was 33+/-10 nmol/g-skeletal muscle.

New multimeric magnetic resonance imaging agents.

RATIONALE AND OBJECTIVES: The authors investigated the effect of multimerization on the relaxivity of macrocyclic gadolinium (Gd) chelates. The objective was to develop more sensitive magnetic resonance imaging (MRI) contrast agents to study biochemical processes. METHODS: Covalently linked nonionic, macrocyclic, multimeric lanthanide chelates that belong to the classes of dimers, trimers, tetramers, hexamer, and octamer, in the molecular weight range approximately 1 to 5 KDa, were synthesized. The chemical linkage was based on either the amide bond or the 2-hydroxypropylidene bond. Relaxivity values, 20r1, on Gd3+ chelates and hydration numbers, Q, on Tb3+ chelates were determined. RESULTS: Relaxivity values increased with molecular weight and Q values were not affected, the increase in r1 in attributable to the expected increase in the overall rotational correlation time, tau r with an increase in molecular weight. The rigidity of the linkers, which is expected to affect the intrachelate rotational correlation time tau r* that makes a contribution to the overall correlation time, tau r, exerted a noticeable effect. The hydroxyl-based chelates generally had lower r1 values than the amide-based chelates. This is rationalized as arising from the longer and thereby rate-limiting effect of the tau m value for the hydroxyl chelates compared with that reported of the amide-based chelates. This rate limiting effect of tau m becomes a dominant factor controlling attainable enhanced relaxivity when multimers based on traditional chelate designs are used for MRI applications. CONCLUSIONS: Approaches aimed at enhancing relaxivity by modulating the water relaxation time, tau m, will be important for the future development of functional MRI contrast agents for the imaging of biochemical processes.

A noninvasive method for monitoring renal status at bedside.

RATIONALE AND OBJECTIVES: The authors demonstrate the feasibility of monitoring renal status continuously and noninvasively at a patient's bedside, avoiding both radioactivity and blood and urine samples. METHODS: Gadolinium-153-labeled ProHance and a glomerular filtration rate (GFR) standard technetium-99m-DTPA were coadministered to anesthetized normal and nephrectomized rats with their tails hanging in a PC 20 spin analyzer. Blood samples and T1 measurements were collected and analyzed. RESULTS: Log time plots of 153Gd, 99mTc (from blood samples) and T1 of the rat tails were all linear and parallel. Halftimes were 32 +/- 2, 32 +/- 6, and 32 +/- 6 minutes for the decay of the T1, 153Gd and 99mTc, respectively. The halftime of the nephrectomized animal was 2000 +/- 4000 minutes. CONCLUSIONS: T1 of an appendage remote from the kidneys reflects the concentration of gadolinium in the blood, which is in rapid equilibrium with tissue interstitial space gadolinium. The decay in T1 of the appendage reflects glomerular filtration. Thus, it is feasible to detect changes in renal status at a patient's bedside by monitoring T1 of a finger or wrist using a small, inexpensive magnet.

Can receptors be imaged with MRI agents?

A review of the feasibility of imaging receptors was made using published literature. The results suggest that there is no physical limitation to imaging the classical biochemical receptors using currently available compounds. Limitations occur because of biological constraints. These constraints are those of delivery of contrast material to the site of the receptor in sufficient quantities and the biological implications of saturating receptors. These limitations are reduced by improving the physics through increasing the relaxivity of the contrast agent either by greater intrinsic relaxivity or by attaching many relaxing agents to the ligand. Biochemical constraints are reduced by targeting receptors involved with transport systems such as receptor mediated endocytosis and by targeting those sites that are in or readily accessible to the vascular system. Once targeted MRI agents are developed their clinical use will, most probably, be different than that of the corresponding radiopharmaceuticals.

Alteration of electronic relaxation in MR contrast agents through de-novo ligand design.

The longitudinal electronic state lifetime of the paramagnetic Gd metal within a macrocyclic ligand core can be increased by designing ligand frames that alter the vibronic interactions between the ligating atoms and the metal. We conducted the first pulsed EPR studies that demonstrated the increase in the longitudinal state lifetimes of the electronic subsystem at cryogenic temperatures. We also designed a simple sucrose/ water model that significantly increases the rotational correlation time in solution of the Gd chelate. This model system enables relaxivity studies at ambient temperatures that more readily interrogate exchange and electronic contributions to the inner-sphere relaxivity by effectively removing the rotational correlation time contribution. These results combined with water residence (Q) measurements suggest that rigidification of the macrocyclic core or that of the pendant arms increases the longitudinal electronic state lifetime of the paramagnetic Gd metal. This increased lifetime possibly contributes to the improved relaxivity for the rigid Gd chelates observed in the sucrose/ water model studies.

The ProHance story: the making of a novel MRI contrast agent.

The four gadolinium chelates currently in clinical use as magnetic resonance imaging (MRI) contrast agents differ in two structural features: linear vs. macrocyclic cores, and ionic vs. nonionic charge types. While all are equivalent in relaxation effectiveness, the nonionic molecules have lower osmolality and viscosity and may be formulated safely at greater concentrations, and delivered confidently at greater doses and as a faster bolus. The macrocyclic molecules are more stable and show less tendency to dissociate free Gd. ProHance was conceived well over a decade ago, based upon a unique structure. It was first marketed in the USA in 1992, and was the first nonionic agent. It remains today still the only commercial MRI agent that is both macrocyclic and nonionic. To date it has been used safely in over a million patients.

Design of conformationally rigid dimeric MRI agents.

The conformational flexibility of the linkage mechanism or tether between paramagnetic metal centers is explored to assess optimal structural characteristics of multimeric MRI contrast agents. Two dimeric paramagnetic chelates differing in linkage flexibility but similar in symmetry are compared Relaxivity, water hydration number, and reorientational times of the chelate groups are obtained, and the results demonstrate that greater linkage flexibility reduces overall relaxivity.

Biodistribution of radiolabeled, formulated gadopentetate, gadoteridol, gadoterate, and gadodiamide in mice and rats.

RATIONALE AND OBJECTIVES: The authors studied the long-term distribution of gadolinium (Gd) in mice and rats after the administration of four commercially available magnetic resonance imaging contrast media. The goals were to determine any possible product dissociation in vivo and to evaluate the effects that product excipients had on the tissue distributions. METHODS: Gadolinium-153 (153Gd)-labeled gadopentetate (Magnevist), gadoteridol (ProHance), gadoterate (Dotarem), and gadodiamide (Omniscan) were administered intravenously to mice (0.48 mmol/kg) and rats (0.1 mmol/kg). At various times up to 14 days posttreatment, the residual 153Gd was measured in selected tissues. The tissue distributions obtained were used to make intra- and interchelate distribution evaluations and comparisons regarding tissue clearance and any possible in vivo dissociation of the Gd chelates. RESULTS: Differences were found among the chelates studied relative to the amounts of residual 153Gd present in tissues known to sequester free Gd, particularly in liver and femur at 7 and 14 days after administration, in both mice and rats. The pattern of the 153Gd distribution suggested that the linear chelates, gadopentetate and gadodiamide, dissociated in vivo resulting in more 153Gd present in bone and liver at the longer residence times than in the subjects injected with the macrocyclic chelates, gadoteridol and gadoterate. The only excipient found to affect the distribution profile was calcium(DTPA-BMA); this excipient in formulated gadodiamide decreased the amounts of residual Gd measured in whole body, bone, and liver in mice compared with levels obtained when gadodiamide was injected alone. CONCLUSIONS: The molecular feature found to be most important in differentiating the four chelates evaluated is the presence or absence of a macrocyclic structure. The Gd chelates containing this structure, gadoteridol and gadoterate, have the lowest residual Gd at long residence times in both mice and rats. The order of residual whole body Gd at 14 days (lowest to highest) was: gadoteridol integral of gadoterate < or = gadopentetate << gadodiamide. The only excipient that affected the biodistribution was found in the gadodiamide formulation where the addition of 5% calcium (DTPA-BMA) reduced residual Gd to just less than 10 times greater than that found for the macrocyclic chelates with the lowest residual Gd, gadoteridol and gadoterate.

MRI evaluation of potential gastrointestinal contrast media.

Diluted ProHance [Gd(HP-DO3A), Squibb Diagnostics, Princeton, NJ], Sustacal (Meadjohnson, Evansville, IN), a nutritional drink, and a ProHance/Sustacal mixture have been investigated as potential oral contrast agents. At 2 T, T1-weighted (SE 500/20) images demonstrated hyperintense (positive) signal enhancement of rat GI tracts within 10 min after the ingestion of 2.0 mM Gd(HP-DO3A) or 2.0 mM ProHance/Sustacal. T2-weighted (SE 3000/80) images demonstrated hypointense (negative) signal intensity within 10 min after ingestion of 10 mM ProHance. Medical imaging applications of these oral contrast media are feasible.