Peptide optimization and conjugation strategies in the development of molecularly targeted magnetic resonance imaging contrast agents.

Peptides are highly selective, high-affinity ligands for a diverse array of disease targets, but suitably derivatizing them for application as diagnostic or therapeutic agents often presents a significant challenge. Covalent modification with metal chelates frequently results in decreased binding affinity, so a variety of strategies must be explored to find suitable locations for modification and facile peptide conjugation chemistries that maintain or enhance binding affinity. In this chapter, we present a paradigm for systematically optimizing peptide binding and determining the favorable sites and methods for peptide conjugation. This strategy is illustrated by two case studies of peptide-based targeted gadolinium contrast agents: EP-2104R for diagnosis of thrombosis and EP-3533 for diagnosis of cardiac perfusion and fibrosis. Two different architectures for the peptide-metal complex conjugation were designed: EP-2104R contains a total of four gadolinium (Gd) chelates linked at the N- and C-termini, whereas EP-3533 is derivatized with three Gd chelates, two on the N-terminus and one on a lysine side chain. Detailed protocols are provided for two Gd chelate conjugation methods.

Serotonin genes and gene-gene interactions in borderline personality disorder in a matched case-control study.

Lines of evidence suggest serotonin genes are susceptibility candidates in borderline personality disorder (BPD). However, few molecular genetic studies on BPD have been reported, especially an overall lack of study on epistatic interactions. We genotyped 27 polymorphisms in 7 serotonin genes in 113 Caucasian BPD patients and matched (sex, age and ethnicity) controls. Program UNPHASED was used to perform association analyses for genotypes, alleles and haplotypes with a permutation test of 10,000 simulations. The Multifactor Dimensionality Reduction analysis was used to examine gene-gene interactions in serotonin system, including three other genes (5-HTT, 5-HT2A and MAOA) that we previously reported. Genotype and allele analyses showed that BPD significantly associated with 5-HT2C and TPH2. BPD patients had high frequencies of the 5-HT2C rs6318G allele (p=0.021) and G/G genotype (OR=2.25); and TPH2 rs2171363T allele (p=0.001) and T containing genotypes (OR=3.40). The 5-HT1A, 5-HT1B, 5-HT1D, 5-HT3A and TPH1 showed no significant association with BPD for genotype, allele and haplotype analyses. We also detected significant interactions between 5-HT2C and TPH2 (p=0.001), and among 5-HT2C, 5-HTT, MAOA and TPH2 (p=0.001) in BPD. Patients with 5-HT2C rs6318G/G genotype had a high frequency of TPH2 rs2171363C/T genotype compared with controls. Our study indicates ""that serotonin genes and their interactions may play a role in the susceptibility to borderline personality disorder.

EP-2104R: a fibrin-specific gadolinium-Based MRI contrast agent for detection of thrombus.

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.

Monoamine oxidase a gene is associated with borderline personality disorder.

OBJECTIVE: Monoamine oxidase A is a mitochondrial enzyme involved in the degradation of certain neurotransmitter amines: serotonin and norepinephrine. As for its role in aggression, impulsivity, suicide and mood liability, monoamine oxidase A can be considered a functional candidate in borderline personality disorder. METHODS: To test for this hypothesis we genotyped two polymorphic markers in monoamine oxidase A gene, a promoter VNTR and an rs6323 (T941G) in exon 8, in 111 Caucasian borderline personality disorder patients and 289 Caucasian healthy controls. Association analyses using individual marker and haplotype data were performed by a program of COCAPHASE in UNPHASED (MRC Human Genome Mapping Project Resource Centre, Cambridge, UK). RESULTS: We found that the borderline personality disorder patients had a high frequency of the high activity VNTR alleles (chi=4.696, P=0.03) and a low frequency of the low activity haplotype (chi=5.089, P=0.02). CONCLUSION: These results show that the monoamine oxidase A gene may play an important role in the etiological development of the borderline personality disorder.

Serotonin 2A receptor gene is associated with personality traits, but not to disorder, in patients with borderline personality disorder.

Borderline personality disorder (BPD) is a chronic, disabling, and high-risk mental disorder characterized by a pervasive pattern of instability in regulation of emotion, interpersonal relationships, self-image, and impulse control beginning in early adulthood. BPD affects about 1%-2% of the general population and has a high mortality rate as a result of suicide and impulsive behaviour. The serotonin 2A receptor gene (HTR2A) is considered a candidate gene for BPD because multiple lines of evidence suggest that it plays an important role in suicide, impulsivity and emotional liability. To test for an association between HTR2A and BPD, we genotyped four polymorphisms, rs6313 (T102C), rs4941573, rs2296972 and rs6314 (His452Tyr), in 111 Caucasian patients with BPD and 287 Caucasian healthy controls. The program UNPHASED was used to compare allele and haplotype frequencies between cases and controls. We did not find a significant association between HTR2A and BPD based on allele, genotype or haplotype analyses. However, there were significant associations between HTR2A and personality traits in the BPD patients. The C allele of rs6313 and the A allele of rs4941573 associated with a higher Extraversion score. Our results suggest that the serotonin 2A receptor gene may not play a major role in the aetiology of borderline personality disorder, but may have a role in personality traits.

Association between serotonin transporter gene and borderline personality disorder.

Borderline personality disorder (BPD) is characterized by a pervasive pattern of instability in regulation of emotion, interpersonal relationships, self-image, and impulse control beginning in early adulthood. BPD affects about 1-2% of the general population and has a high mortality rate as a result of suicide and impulsive behaviour. The serotonin transporter gene (5-HTT) is considered as a candidate gene for BPD as multiple lines of evidence have suggested that it plays an important role in suicide, impulsive behaviour, and emotional liability. To test for an association between 5-HTT and BPD, we genotyped three common polymorphisms: the serotonin transporter linked promoter region (5-HTTLPR); a variable number of tandem repeat (VNTR) in intron 2, and a single nucleotide variant (A/G) within the LPR region. Eighty-nine Caucasian patients with BPD and 269 Caucasian healthy controls were analyzed. The program UNPHASED was used to compare allele and haplotype frequencies between cases and controls. Significant differences in allele frequencies of the VNTR marker (p=0.012) and haplotype frequencies (p=0.002) between patients and controls were found. Compared with healthy controls, patients with BPD showed higher frequencies of the 10 repeat of the VNTR marker and the S-10 haplotype, and lower 12 repeat and L(A)-12 haplotype. Our results suggest that the serotonin transporter gene may play a role in the aetiology of borderline personality disorder.