New York University School of Medicine Drug Development Educational Program: 2-Year Benchmark.

Drug development (DD) is a multidisciplinary process that spans the translational continuum, yet remains an understudied entity in medical schools and biomedical science institutes. In response to a growing interest and unmet need, we implemented a DD course series that details identification of viable molecular targets, clinical trial design, intellectual property, and marketing. Enrollment is open to faculty, postdoctoral trainees, and MD, PhD, and MS students. After 2 years, 37 students and 23 students completed the fall and spring courses, respectively. Pre/post-surveys demonstrated gained knowledge across course topics, with mean survey scores increased by 66% (p < 0.001) after each course. Lectures for each course were consistently rated highly, with a mean course rating of 4.1/5. Through this program, trainees will have a more innovative approach toward identification of therapeutic targets and modalities. Furthermore, they will learn to integrate technology and biomedical informatics to find creative solutions in the DD process.

Microvascular angina in systemic hypertension: diagnosis and treatment with enalapril.

The causes of hypertensive microvascular ischemia are reviewed along with diagnostic factors. Stress/rest thallium-201 scintigraphy is shown to have a predictive value of 78% for a diagnosis of microvascular disease in hypertensive patients with exertional angina and left ventricular hypertrophy. Lack of isotope uptake at peak stress correlates well with the decrease in coronary flow reserve in ischemic segments, which is 2-3 times lower than in normal subjects. Treatment with enalapril produces regression of left ventricular hypertrophy, normalization of thallium-201 uptake, and an increase in exercise capacity in patients with microvascular angina.

Microvascular angina pectoris in hypertensive patients with left ventricular hypertrophy and diagnostic value of exercise thallium-201 scintigraphy.

In a series of 120 hypertensive patients, 60 were found to have echocardiographic left ventricular (LV) hypertrophy (Devereux's method). Of these, 18 (30%) had typical stress-induced angina and underwent coronary angiography, which showed that 11 (61%) had normal coronary arteries, and 7 (39%) (p < 0.05) had coronary stenosis of the epicardial arteries. Stress-rest thallium-201 scintigraphy (Burow's quantitative method) yielded abnormal results in 21 of the 60 patients with LV hypertrophy. Five of 30 (17%) were asymptomatic, 14 of 18 (78%) had angina, and 2 of 12 (17%) had dyspnea on exertion. In 5 normal patients used as a control group, coronary flow reserve after administration of papaverine (10 coronary arteries) was 6.25 +/- 1.4 versus 3.7 +/- 0.8 in 10 thallium-negative, asymptomatic hypertensive patients with LV hypertrophy (p < 0.001). The mean coronary flow reserve of 21 patients with abnormal thallium-201 results was 2.71 +/- 0.96 (p < 0.01 compared with the group with normal thallium-201 findings) and 2.5 +/- 0.6 in the segments with lowest uptake (p < 0.05 compared with normal segments in these same patients). Thus, stress-induced angina pectoris in hypertensive patients with LV hypertrophy was due to small-vessel disease in over half of our patients (62%).

Biodistribution of the Cuban anti-meningococcal vaccine, VA-MENGOC-BC, in Balb/c mice.

VA-MENGOC-BC is the Cuban vaccine against Neisseria meningitidis BC. Its iodination and biodistribution measurement in Balb/c mice were the main goals of this study. The Chloramine-T method was effective for radiolabelling the proteoliposome, the main vaccine structure. The biodistribution demonstrated that the thyroid (50.4%), muscle (21.5%) and regional lymph nodes (20.5%) were the most radioactive organs and the kinetic of radioactivity was correlated with the primary (muscle, highest values in the first 3 days and lymph nodes, in the first 7 days) and secondary (muscle and lymph nodes, highest values in the first day) response. Early occurrence of slight radioactivity in the spleen was also observed. This was the first time that the iodination and biodistribution of this vaccine was carried out. The present study corroborated that the time selected in previous trials to obtain the lymph nodes and spleen cell, after a first (7 days) and second (3 days) dose, was actually the optimal for in vitro studies.

Antithrombin: structure, genomic organization, function and inherited deficiency.

Antithrombin is a major plasma protein inhibitor of proteinases generated during blood coagulation; it plays an important role in the regulation of thrombin in blood. The anticoagulant heparin greatly accelerates the rate of inactivation of proteinases by antithrombin, predominantly through its well defined, highly specific binding reaction with the inhibitor, but also through a less strictly defined interaction with some of the proteinases (such as thrombin). There is evidence for an analogous acceleratory mechanism in vivo, that functions by the binding of antithrombin to a subpopulation of heparan sulphate proteoglycans intercalated in the surface of endothelial cells. The location and structure of the gene for antithrombin are known. Both its overall organization and the structure of the subdomains of the expressed protein can be considered in terms of their relationships to a serine proteinase inhibitor superfamily, which is believed to have evolved from a common ancestor. The region of the antithrombin gene 5' to the coding region has been characterized. Unlike other members of the serpin family, there is no TATA-like promoter sequence. Two enhancer sequences have been identified that are homologous to enhancer regions of other genes. There are two polymorphisms: an intragenic polymorphism arising from a translationally silent A to G transition in codon 305, and a length polymorphism arising from the presence of 32 bp or 108 bp non-homologous sequences 345 bp upstream from the translation initiation codon. Inherited deficiency of antithrombin is associated with familial thromboembolism. The molecular genetic basis of some subtypes of deficiency is increasingly yielding to investigation. It is interesting to note that a number of mutations have been identified in CpG dinucleotides, supporting the suggestion that this dinucleotide sequence may represent a mutation hotspot in the human genome.

Permanent pacemaker implantation using the femoral vein: a preliminary report.

A permanent pacemaker was implanted through the femoral vein in 23 patients using the percutaneous puncture technique. The pulse generator was placed in the lower abdominal wall. The method is simple and reduces the time necessary to accomplish implantation. Catheter extrusion in one patient was easily corrected. Another patient had late thrombophlebitis, possibly unrelated to the procedure. Catheter dislodgement occurred in four (4) patients and penetration of the right atrial appendage and right ventricular apex each occurred once. We believe these problems can be circumvented with more experience and expect the femoral approach to be a simple and practical method permanent pacemaker implantation.

Functional and organic components of diastolic dysfunction in hypertensive heart disease: therapeutic implications.

The major structural and functional determinants of impaired left ventricular diastolic function in the hypertensive patient are reviewed, together with the indices normally used to detect this failure. The alteration of functional determinants can be quickly modified, while structural determinants are modified only over the long term. Drug therapy first affects the functional determinants, bringing about their attenuation and initiating the modification of the structural factors, thus accounting for the improvement in diastolic function over the long term.

Segmentary coronary reserve in hypertensive patients with echocardiographic left ventricular hypertrophy, gamma-graphic ischaemia and normal coronary angiography.

Stress thallium scintigraphies are frequently positive in patients with systemic hypertension (SHT), especially in the presence of left ventricular hypertrophy (LVH). In order to determine whether positive thallium perfusion scans in patients with LVH secondary to SHT and normal coronary angiographies are due to segmentary reduction of coronary reserve (CR), we have studied 10 out of 60 consecutive cases of SHT with echocardiographic LVH, using intracoronary Doppler. We compared coronary blood flow velocity at rest and post-papaverine (PP), and CR in at least two major coronary vessels, always including the one corresponding to the ischaemic segment. In the vessel with the least CR at rest, a new determination of CR was made under intracoronary nitroglycerin. A group of five normal patients acted as controls. The mean CR of the controls and patients, respectively, was 6.2 +/- 1.4 vs 2.7 +/- 0.9 (P < 0.001). In patients with positive thallium perfusion scans, the coronary arteries corresponding to the ischaemic segments had less CR (2.5 +/- 0.6) than arteries from non-ischaemic segments (3.4 +/- 1, P < 0.05). These differences were greater when the ischaemia was anterior. There was no correlation either between CR and left ventricular mass (r = 0.23) or rest coronary blood flow velocity (r = 0.07). Only one patient exhibited functional behaviour indicating reduced CR; this rose from 1.9 to 7.5 after nitroglycerin 300 micrograms. In conclusion, CR determined by intracoronary Doppler and papaverine shows segmentary differences both in normal patients and in patients with LVH and normal coronary angiograms. This could be the cause of segmental ischaemia detected by means of radionuclide stress tests.

Important role of arginine 129 in heparin-binding site of antithrombin III. Identification of a novel mutation arginine 129 to glutamine.

An hereditary abnormal antithrombin III (ATIII Geneva) with defective heparin cofactor activity was characterized by DNA single strand amplification and subsequent direct sequencing. ATIII Geneva was found to have a G to A transition in Exon IIIa leading to an Arg-129 to Gln mutation. This amino acid is part of the ATIII region comprising residues 114-154, which contains the highest proportion of basic residues (Arg or Lys), and is known from chemical modification studies to be involved in heparin binding. The variant protein did not bind heparin-Sepharose and was isolated from the propositus plasma by immunoaffinity chromatography. High affinity (for ATIII) heparin had only a minimal effect on thrombin and activated factor X inhibition by the purified abnormal ATIII. Taken together, these results demonstrate an important role for Arg-129 in the binding and interaction of ATIII with heparin of high affinity. We propose that a cooperation between Lys-125, Arg-129, Lys-136, and Arg-47 exposed at the surface of the inhibitor allows the binding of the essential pentasaccharide domain of heparin which is specific for the ATIII interaction.

Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family.

Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin-Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

Antithrombin Vicenza, Ala 384 to Pro (GCA to CCA) mutation, transforming the inhibitor into a substrate.

Antithrombin (AT) Vicenza has been previously identified as a functionally abnormal antithrombin associated with familial thrombosis (Finazzi et al, 1985). It binds normally to heparin, but loses its affinity following interaction with thrombin: it is a poor inhibitor of thrombin. AT Vicenza was isolated from plasma by heparin-Sepharose and thrombin-Sepharose chromatography, fragmented with cyanogen bromide (CNBr) and its tryptic peptides were analysed by fast atom bombardment mass spectrometry mapping. An abnormal peptide mass 1112 was identified. Edman degradation confirmed a substitution of Ala to Pro in the sequence Ala 383-Arg 393. Polymerase chain reaction amplification of exon 6 of the gene followed by genomic sequencing, localized the mutation to codon 384, GCA to CCA. The same mutation has recently been reported in AT Charleville (Mohlo-Sabatier et al, 1989). Sodium dodecyl-sulphate polyacrylamide gel electrophoresis of AT Vicenza (/Charleville) under non-reducing conditions revealed an apparent increase in mol. wt following interaction with thrombin: under reducing conditions the mol. wt was less than that of normal AT. This indicated cleavage and unfolding of the molecule. The site of cleavage was determined by incubation of AT Vicenza (/Charleville) with thrombin-Sepharose, reduction and S-carboxymethylation and reverse phase FPLC. A peptide was identified with the NH2-terminal sequence beginning Ser-Leu-Asn, demonstrating the cleavage had occurred at the reactive site of the variant. It is concluded that the Ala 384 to Pro substitution transforms AT Vicenza (/Charleville) from an inhibitor into a substrate.