A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis.

The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.

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.

Methyl methacrylate: inhalation developmental toxicity study in rats.

Methyl methacrylate (99.9% pure) was administered by vapor inhalation exposure to five groups (27 rats/group) of presumed pregnant rats (Crl:CD) at concentrations of 0 (control), 99, 304, 1,178, and 2,028 ppm for 6 hr/day on days 6-15 of gestation (G). Maternal body weight, feed consumption, and clinical signs were recorded throughout gestation. Dams were euthanized on day 20 G. Each uterus was weighed and corpora lutea, implantation sites and resorptions were counted. The number of fetuses per litter were counted and their location within the uterus recorded. All fetuses were weighed, sexed and examined for external and skeletal alterations. One half of the fetuses from each litter were examined for visceral alterations. No treatment-related deaths were noted at any concentration tested. Treatment-related effects on maternal body weight and feed consumption were noted at all exposure levels. The decreases in maternal body weight at 99 and 304 ppm were minimal and transient since they returned to control values by the next weighing period. When exposure was discontinued, body weight gain and feed consumption in all exposure groups returned to control values. There were no treatment-related changes in the number of litters produced or in the mean number per litter of corpora lutea, implantations, resorptions, live or dead fetuses, or sex ratio. Fetal body weights were similar between the control and treated groups. There were no treatment-related increases in the type or incidence of external, visceral, or skeletal malformations, developmental variations, or variations indicative of retarded development. Exposure to methyl methacrylate concentrations up to 2,028 ppm resulted in no embryo or fetal toxicity or malformations even at exposure levels that resulted in maternal toxicity.

Effect of N-methyl substitution of the peptide bonds in luteinizing hormone-releasing hormone agonists.

Each peptide bond in leuprolide (1), deslorelin (13), and nafarelin (24) was separately substituted with N-methyl. The synthesized compounds were tested for in vitro receptor binding, LH release, and stability against chymotrypsin and intestinal degradation. The NMe-Ser4 (30), NMe-Leu7 (33), and Sar10 (35) analogues of nafarelin had pD2 values 2-, 20-, 9-fold higher than their respective parent. All the other N-methyl agonists were less active. For the first time, conversion of LHRH agonists to antagonists was observed as a result of N-methyl substitution in the peptide backbone. [NMe-Phe2,DLeu6,Pro9NHEt]LHRH (4), [NMe-1Nal3,DLeu6,Pro9NHEt]LHRH (6), [NMe-His2,DTrp6,Pro9NHEt]LHRH (14), [NMe-Phe2,DNal6]LHRH (27), and [D2Nal6,NMe-Arg8]LHRH (34) exhibited antagonist responses. Substitutions of NMe-1Nal3, NMe-Ser4, or NMe-Tyr5 in leuprolide rendered the 3-4 peptide bond in these compounds completely stable to chymotrypsin. Examination of the three-dimensional structure of leuprolide when bound to the active site of chymotrypsin, reveals the NH's of residues 3 and 5 are involved in hydrogen bond interactions with the enzyme. N-Methylation at these positions is not only disrupting the hydrogen bond interactions, but is also sterically preventing the substrate from fitting in the enzyme's active site. All the compounds in the leuprolide series were also tested against intestinal degradation using an in vitro rat jejunum sac assay. In this model the pattern of stabilization was similar, but not identical, to that against chymotrypsin. The pharmacokinetics of all the analogues in the leuprolide series and of several others in the deslorelin and nafarelin series were determined. The clearance values of all the three NMe-Tyr5 analogues, 8, 20, and 31 were lower than their respective parents. These slower clearances suggest lower rates of metabolism.

Stabilization of the N-terminal residues of luteinizing hormone-releasing hormone agonists and the effect on pharmacokinetics.

To stabilize leuprolide (1) against chymotrypsin and intestinal degradation several agonists of LHRH (2-12), modified at position 1, 2, or 3 and/or containing N-alpha-methyl at positions 1, 2, or 4, were synthesized by SPPS. These agonists were tested in vitro for (a) rat pituitary LHRH receptor binding, (b) LH release from rat pituitary cells, (c) stability against chymotrypsin, and (d) stability against rat intestinal degradation. The clearances of the compounds in the rat were determined using a RIA. Complete stabilization against chymotrypsin (t1/2) and lumenal degradation (T1/2) was achieved with substitution of NMe-Ser4 in leuprolide; however, with an increase in clearance. Substitution with 1-Nal3 increased both t1/2 and T1/2, while substitution with NAc-Sar1 increased only T1/2. [NAcSar1,NMeSer4,D-Trp6,Pro9NHEt]LHRH (12), the doubly stabilized analogue, was tested in the rat by both iv and id administrations, and its bioavailabilities were measured. No significant improvement in id absorption over leuprolide was observed.

Apudoma of the pancreas: benign or malignant?

A middle-aged, obese, black woman complained of abdominal pain and tenderness in the left upper quadrant. An abdominal computerized tomographic (CT) scan revealed an encapsulated cystic mass in the tail region of the pancreas. Selective angiography confirmed mass and hypervascularity, definite encapsulation, and lack of capsular invasion. The diagnostic value of the CT scan, angiography, and special staining in classifying the excised pancreatic mass as an apudoma is discussed. Malignancy was excluded by the lack of capsular-vascular invasion and the absence of metastases. Nonfunctional status was determined by lack of hormone hypersecretion.

The obtuse angle tongue depressor: physiologic advantages in throat surgery and lesions of the hypopharynx.

The original purpose of the obtuse angle tongue depressor was to open the mouth much wider than the right angle tongue depressor and to expose the lower pole of the tonsil for more complete surgical treatment. However, the later discovery of the physiologic advantages of the obtuse angle tongue blade in opening the mouth became very important. The obtuse angle tongue blade pulls the tongue forward so that benign lesions can be excised as well as adequate biopsies of malignant tumors obtained in most of the hypopharynx. The clinical finding of the small amount of pressure on any part of the tongue when the obtuse angle tongue depressor is used indicates minimum trauma to the patient.