Potent and highly selective hypoxia-activated achiral phosphoramidate mustards as anticancer drugs.

A series of achiral hypoxia-activated prodrugs were synthesized on the basis of the DNA cross-linking toxin of the prodrug, ifosfamide. The hypoxia-selective cytotoxicity of several of the compounds was improved over previously reported racemic mixtures of chiral bioreductive phosphoramidate prodrugs. Prodrugs activated by 2-nitroimidazole reduction demonstrated up to 400-fold enhanced cytotoxicity toward H460 cells in culture under hypoxia versus their potency under aerobic conditions. Compounds were further assessed for their stability to cytochrome P450 metabolism using a liver microsome assay. The 2-nitroimidazole containing lead compound 3b (TH-302) was selectively potent under hypoxia and stable to liver microsomes. It was active in an in vivo MIA PaCa-2 pancreatic cancer orthotopic xenograft model as a monotherapy and demonstrated dramatic efficacy when used in combination with gemcitabine, extending survival with one of eight animals tumor free at day-44. Compound 3b has emerged as a promising antitumor agent that shows excellent in vivo efficacy and is currently being evaluated in the clinic.

Age-Associated Changes in Parameters of Left Ventricular Diastolic Function Assessed by Pulsed Doppler Myocardial Imaging of Mitral Annulus in Healthy Korean Adults

BACKGROUND AND OBJECTIVES: Left ventricular (LV) diastolic function can be evaluated by obtaining parameters from Doppler analysis of the mitral inflow, which are known to change with increase in age. Pulsed Doppler myocardial imaging (DMI) analysis of mitral annular velocity has been proposed as a more accurate method for evaluation of LV diastolic function. This study sought to find out the age-associated changes in parameters of LV diastolic function acquired from pulsed DMI analysis of the mitral annulus in a large group of heathy Korean adults. MATERIALS AND METHODS: Two hundred and eighty-nine apparently healthy Korean subjects who visited the Center for Health Promotion for routine health checkup were studied. All went through pulsed DMI by echcardiography for acquisition of velocities in the septal, lateral, anterior, and inferior annuli of the mitral valve. Peak early diastolic velocity (Em), peak late diastolic velocity (Am), and their ratio (Em/Am) were obtained and their correlation with age was evaluated. Doppler flow analysis of mitral inflow was also performed. Peak early and late diastolic flow velocity (E and A), deceleration time (DT) and isovolumic relaxation time (IVRT) were assessed and their relationship with increase in age was also evaluated. RESULTS: Mean age of the subjects was 55.7+/-10.9. Em velocities of all portions of mitral annulus showed good correlation with age, showing continuous decline with increase in age (R=-0.60, -0.58, -0.59, -0.58 for septal, lateral, anterior and inferior annuli, respectively, p<0.01). Am velocities showed a significant but minimal increase with increase in age in all of the sampled positions. Em/Am ratio also showed a significant decline similar to the change in Em velocities. The A velocity and E/A ratio obtained from mitral inflow Doppler analysis showed a significant decline and DT and IVRT showed a significant increase with increase in age. CONCLUSION: Parameters of left ventricular diastolic function evaluated by pulsed DMI show significant changes that correlate well with increase in age. This supports the finding that left ventricular diastolic function continuously declines with increase in age.

The Change of Expression of Troponin T and I Isoforms in the Failing Myocardium of Rat after Myocardial Infarction

BACKGROUND: Contractile dysfunction of rat myocardium in postinfarction remodeling is characterized by decreased response of myofilament both to calcium(Ca++) and adrenergic stimuli. This study tested the hypothesis that above alterations may be linked to molecular switches among the isoforms of troponin T and troponin I, the major regulators of myocardial responsiveness. METHODS: Using Sprague-Dawley rat as a model, myocardiums of fetal heart, 1 day postnatal heart, normal adult heart(n=4), and non-infarcted area of postinfarction heart(n=4, 3 months after left coronary artery ligation, mean LVEDP=21.4mmHg) were studied. For the detection on molecular switches, western blotting and semiquantitative RT-PCR were employed. RESULTS: Immunoblotting of rat myocardium showed normal developmental isoform switch of troponin protein. There was distinct isoform patterns specific for postinfarction rat heart. The postinfarction myocardium developed a marked increase in the fetal isoform and marked decrease in the adult isoform of troponin T, resulting in the reversed ratio of fetal/adult cardiac troponin T isoform(normal adult rat=0.60+/-0.1 vs postinfarction rat=1.79+/-0.15, p<0.01). Also, the postinfarction heart showed approximately 20% decrease in the amount of adult troponin I isoform. In RT-PCR experiments, the postinfarction hearts were characterized by increased amplification of fetal troponin R isoform cDNA(fetal troponin R/GAPDH ; normal adult rat=0.22, postinfarction rat=0.84). However, there was no siginificant difference in the amplification of troponin I cDNA between normal and postinfarction heart. CONCLUSION: These experimental findings indicate that there are molecular switches operative among the regulatory protein troponin T and I in the rat myocardium with development of postianfarction heart failure.

Induction of Embryonal Teratocarcinoma Cell(P19 cell) to Differentiate into Smooth Muscle Cell

BACKGROUND: Understanding the regulatory mechanisms of the smooth muscle cells differentiation is one of the central issues in researches of atherogenesis where smooth muscle cells undergo dedifferentiation and regain embryonic phenotype. Smooth muscle myosin heavy chain isoforms(SM1,SM2) are important molecular markers to define the stage of smooth muscle cell differentiation. METHODS: In order to establish an in vitro model of smooth muscle cell differentiation using a pluripotent murine embryonal teratocarcinoma cell line(P19 cell), we first isolated cDNA clone of mouse SM1 and then tried various chemicals to induce P19 cells to differentiate into smooth muscle cells, The expression of Sm1 and alpha-smooth muscle actin was examined using RNase protection assay, Western blotting and indirect immunofluorescence. RESULTS: In the presence of luM retinoic acid, a small proportion of P19 cells could be in duced to differentiate into smooth muscle cells expressing SM1 as well as alpha-smooth muscle actin since day 8 after treatment. By blocking the Brain-2 expression, thus inhibiting neuronal differentiation, we could obtain more abundant differentiated smooth muscle cells. Sequential immunofluorescencc demonstrated that it took weveral days for smooth muscle myosin heavy chain to organize completely in differentiation smooth muscle cells. On the other hand, 10nM retinoic acid, 1% dimethyl sulfoxide or 2mM hexamethylene bisacetamide could not indduce P19 cell to differentiate to smooth muscle cells. CONCLUSION: In conclusion, P19 cells can be induced to differentiate into smooth muscle cells and this in vitro system will be useful in understanding the regulation of SM1 gene expression as well as of angiogenesis.

A Study of the Cumulative Effect of the Postischemic Prolonged Myocardial Dysfunction by Repeated Coronary Artery Occlusion and Reperfusion

In order to observe the changes in left ventricular systolic and diastolic function by repetitive regional myocardial ischemia and reperfusion, left anterior descending artery(LAD) just distal to the first diagonal branch in nine mongrel dogs was obstructed for 1 minute and reperfused for 15 minutes twice (first and second obstruction and reperfusion), and as a third trial repeated obstruction for 5 minutes and reperfusion for 15 minutes was done. Then the last obstruction for 1 minute and reperfusion for 15 mintues was performed. Peak positive dp/dt, left ventricular systolic pressure(LVSP) and systolic thickening were used as systolic parameters and peak negative dp/dt, left ventricular end diastolic pressure (LVEDP) and time constant of isovolumic relaxation were used as diastolic parameters. The results are as follows : 1) After first reperfusion, all systolic and diastolic parameters showed no significant changes except systolic thickening, which fell from the basal line(48.9+/-16.6% versus 59.8+/-10.0%, p<0.05). 2) After second reperfusion, peak positive dp/dt and systolic thickening decreased (1475+/-342mmHG/sec versus 1561+/-307mmHg/sec,p<0.05; 48.2+/-6.1% versus 59.8+/-10.05%, p<0.005 respectively). But LSVP, LVEDP, peak negative dp/dt and T showed no significant changes. 3) After third reperfusion all systolic and diastolic parameters except LVEDP showed significant impairment by 5 minutes of coronary occlusion, which means postischemic prolonged myocardial dysfunction(peak positive dp/dt 1401+/-362mmHg/sec versus 1561+/-307mmHg/sec, p<0.005; LVSP 88.5+/-23.4mmHg versus 97.6+/-25.4mmHg, p<0.05; systolic thickening 41.2+/-8.2% versus 59.8+/-10.0%, p<0.005; peak negative dp/dt -879+/-299mmHg/sec versus -1037+/-297mmHg/sec, p<0.05; T 46.3+/-8.2 msec versus 41.9+/-6.1 msec, p<0.01). 4) All observed systolic and diastolic parameters also revealed myocardial dysfunction after fourth reperfusion(peak positive dp/dt 1348+/-288mmHg/sec versus 1561+/-307mmHg/sec, p<0.0005; LVSP 88.5+/-24.1mmHg versus 97.6+/-25.4mmHg, p<0.005;systolic thickening 32.1+/-8.9 versus 59.8+/-10.0%, p<0.001; peak negative dp/dt -850+/-260mmHg/sec versus -1037+/-297mmHg, p<0.05; LVEDP 6.6+/-1.7mmHg versus 5.4+/-1.3mmHg, p<0.05; T 49.9+/-9.8msec versus 41.9+/-6.1msec, p<0.01). 5) Overall tendencies of myocardidal impairment were statistically significant in each parameter (peak positive dp/dt, p<0.001; systolic thickening, p<0.05; LVEDP, p<0.05; T, p<0.01) except LVSP and peak negative dp/dt. Thus myocardial function was impaired progressively by repetitive regional myocardial ischemia and reperfusion. In conclusion, a 5 minute coronary occlusion results in postischemic prolonged myocardial ischemia and repetitive coronary occlusion and reperfusion may show cumulative effect.

Assessment of Left Ventricular Diastolic Function and Effect of Calcium Channel-Blocking Agent on Diastolic Function by Pulsed Doppler Echocardiography in Patients with Hypertrophic Cardiomyopathy

The symptoms of hypertrophic cardiomyopaty frequently result from impaired left ventricular relaxation, abnormal left ventricular filling, and decreased compliance of left ventricle in spite of normal systolic function. Several studies have suggested that the assessment of transmitral flow velocity waveform with pulsed Doppler echocardiography in patients with hypertrophic cardiomyopathy provide a noninvasive and clinically useful expression of left ventricular diastolic performance. In this study, pulsed Doppler echocardiography was used to measure diastolic indices from transmitral flow velocity waveform and thereby to assess left ventricular diastolic function in 20 patients with hypertrophic cardiomyopathy (14 septal hypertrophy, 3 apical hypertrophy, and 3 concentric hypertrophy). The diastolic indices to measure are isovolumic relaxation time(IVRT), deceleration time(DT), pressure half time(PHT), deceleration of early diastolic flow(DEF), EF slope, peak flow velocity in early diastole(PFVE), peak flow velocity during atrial systole(PFVA), and PFVE/PFVA ratio. The diastolic indices obtained from patients were compared with those in 20 age-matched control subjects without heart disease. The relationship between left ventricular wall thickness index(Th Index) and diastolic indices were evaluated. In addition, the effect of calcium channel-blocking agent on left ventricular diastolic function were evaluated. The results were as follows ; 1) There were no significant differences in RR interval, BP, end-systolic left ventricular dimension, and end-diastolic left ventricular dimension, but significant differences in interventricular septal thickness, posterior wall thickness, and left atrial dimension between hypertrophic group and control group. 2) The systolic index(ejection fraction) showed no significant difference between hypertrophic group and control group. 3) All diastolic indices except PFVA showed significant differences between hypertrophic group and control group. 4) Th Index did not showed a significant correlation with the diastolic indices except PFVE/PFVA. 5) There were no significant changes in RR interval, BP end-systolic left ventricular dimension, end-diastolic left ventricular dimension, left atrial dimension, and ejection fraction between medication and drug withdrawal. IVRT significantly increased after drug withdrawal. But other diastolic indices showed no significant changes after drug withdrawal. According to above results the assessment of left ventricular diastolic function by pulsed Doppler echocardiography was a clinically useful method in detecting diastolic dysfunction in patients with hypertrophic cardiomyopathy. Calcium channel-blocking agent may have beneficial effect in improving left ventricular diastolic function in patients with hypertrophic cardiomyopathy.

A Study on Coronary Sinus Occlusion Pressure and the Effect of Coronary Sinus Occlusion on Left Ventricular Function

In order to observe the effect of coronary sinus occlusion on left ventricular function, coronary sinus was occluded for 2 minutes using Swan-Ganz catheter in 9 mongrel dogs. The change in gross findings of heart was observed, and coronary sinus pressure, LV pressure, LV dp/dt, and ECG were recorded throughout the experiment. The coronary sinus occlusion pressure(CSOP) rised slowly reaching to the plateau at the 32+/-6th beat and at 11.5+/-3.5 seconds after coronary sinus occlusion, with the pressure 95.4+/-24.7mmHg in systole and 35.9+/-21.3mmHg in end-diastole. End-diastolic CSOP was different significantly from LV end-diastolic pressure(LVEDP; 6.8+/-3.3mmHg). The veins of left ventricle were congested immediately after coronary sinus occlusion, followed by the dilation of coronary sinus and coronary arteries, and the dilation of left ventricle with visible decrease in its contractility. Above changes recovered slowly after the release of coronary sinus occlusion. LV peak systolic pressure, LV peak dp/dt, and LV peak negative dp/dt were decreased, and relaxation time constant was prolonged significantly 30 seconds after coronary sinus occlusion, and they recovered to those of preocclusion period 30 seconds after release of coronary sinus occlusion. LVEDP was increased significantly 30 seconds after coronary sinus occlusion and returned to that of preocclusion period immediately after release of coronary sinus occlusion. heart rate was decreased 1 minute after coronary sinus occlusion and returned to that of preocclusion period immediately after release of coronary sinus occlusion. As a result, there was significant difference between end-diastolic CSOP and LVEDP in the experiment with mongrel dogs, and it should be reevaluated to estimate LVEDP by end-diastolic CSOP in man. Left ventricular systolic and diastolic function was impaired transiently after coronary sinus occlusion lasting more than 30 seconds.

Noninvasive Evaluation of Left Ventricular Diastolic Function in Patients with Ischemic Heart Disease: Reconstruction of Transmitral Flow Rate and Filling Volume Curve with M-Mode, 2-Dimensional and Doppler Echocardiography

To evaluate the disturbed left ventricular diastolic filling by pulsed Doppler echocardiography in patients with ischemic heart disease who have normal systolic function, 117 subjects (50 angina patients with, 39 myocardial infarction patients with, 28 control subjects without significant coronary arterial narrowing) underwent echocardiographic examination one day before coronary arteriography. Beside analyzing trasmitral flow velocity curve, reconstruction and quantitative analysis of left ventricular filling rate and filling volume curves were made from Doppler trasmitral flow velocity curve, 2-Dimensional mitral annulus diameter and M-Mode mitral valve motion. From reconstructed left ventricular filling rate, filling fraction during early rapid filling or half diastolic rate, ratio of early to atrial peak filling rate, filling fraction during early rapid filling or half diastolic period and diastolic time interval(esp. T1/2 from peak early filling rate to its half valve)were measured. Angina and myocardial infarction group had significantly lower normalized peak early filling rate(4.9+/-0.6, 4.8+/-1.2 vs 6.0+/-1.1 DFV/sec, P<0.005), ratio of early to atrial peak filling rate(103.6+/-29. 4120.6+/-3.5 VS 175.5+/-55.0%, P<0.005), filling fraction during early diastolic period(46.2+/-5.0, 44.4+/-12.6 VS 54.3+/-6.8%, P<0.005) and filling fraction during half diastolic period(56.3+/-5.8, 55.4+/-14.1 VS 66.6+/-7.7%, P<0.005) than those of control group. Angina and myocardial infarction group had significantly higher normalized peak atrial filling rate(4.9+/-1.4, 5.0+/-2.0 VS 3.5+/-0.9 DFV/sec, P<0.005), prologed normalized T1/2 (12.5+/-3.0, 12.0+/-4.0 VS 9.8+/-2.2%, P<0.005) and delayed isovolumic relaxation time(81.7+/-7.8, 95.0+/-13.6 VS 74.3+/-6.9msec, P<0.005) than those of control group. Affecting factors to pseudonormalize left ventricular filling rate and filling volume curves in myocardial infarction group were mitral regurgitation, left ventricular aneurysm and severe impairment of systolic function. In 13 angina group patients who had undergone coronary angioplasty, no difference were found in any noninvasive diastolic filling parameters before and immediately(24 hours and 5 days) after the procedure. Thus, abnormal patterns of left ventricular filling occur in patients with ischemic heart disease and near normal global systolic function. The decreased peak early filling rate and early filling fraction occuring during rapid filling and the increased peak atrial filling rate occuring in late diastolic suggest that the patients with ischemic heart disease have impaired early diastolic filling. These diastolic filling abnormalities are unimproved 24 hour and 5 days after succesful coronary angioplasty. These diastolic filling parameters from left ventricular filling rate and filling volume curves provide useful noninvastive hemodynamic indices for assessment of left ventricular diastolic filling in patients with ischemic heart disease.

Evaluation of Left Ventricular Function Using Force-Interval Relationship

The force-interval relationship of cardiac muscle has been known as not only a fundamental manifestation of beat-to-beat kinetics of intracellular activator calcium which control contractile response but also a potential clinical tool for evaluating cardiac contracile function. In this study were evaluated the force-interval relationship of intact canine left ventricle through mechanical restitution curves by plotting contrctile responses to varying steady state, extrasystolic and postextrasystolic intervals, and compared the force-interval relationships of intact canine left and right ventricles quantitatively. Effects of localized myocardial ischemia on the left ventricular force-interval relationship and relaxtion function were also evaluated 30 minutes after ligating proximal left anterior descending coronary artery through observing contractile and relaxtion responses to various intervals. 1) Mechanical restitution curve of left ventricle showed that left ventricular dp/dt max responses rose stiffly until plateau level with increasing postextrasystolic intervals, then declined with further increment of postextrasystolic intervals. 2) Mechanical restitution curve of left ventricle shifted leftward and upward with shortening of steady state and extrasystolic intervals, which suggest intracellular calcium kinetics during electrical diastole may operate as a mechanism of the force-interval relationship. 3) Steady state contractile responses remained unchanged but maximal contractile responses increased significantly or contractile reserve in intact left ventricle. 4) Normalized force-interval relationships of left and right ventricle were similar quantitatively, which suggest the force-interval relationship is independent of structural factors in intact canine heart. 5) Occlusion of coronary artery lowered absolute values of left ventricular dp/dt max responses to varying postextrasystolic intervals, but didn't show significant changes of normalized dp/dt max responses, which suggest force-interval relationship be also present in spite of localized myocardial ischemia. 6) Responses of normalized left ventricular dp/dt min to varying postextrasystolic intervals were similar to those of normalized dp/dt max but reduced after coronary artery occlusion in the range above 100% dp/dt max response, which may be used for the detection and evaluation of deranged myocardial relaxation in the left ventricle with localized myocardial ischemia.

Effect of KCI Continus Tablet(K-Contin(R)) on Serum K and Uric Acid Level during Hydrochlorotihiazide Therapy

The effects of KCI continus tablet(K-Contin(R)) on the serum level of K and uric acid during hydrochlorothizide therapy(25mg/day) were evaluated in 30 patients with essential hypertensien. The results are as follows : 1) During hydrochlorothiazide therapy(25mg/day), 23% of all patients showed hypopotassemia(<3.5mEq/1) while no patient developed hypopotassemia after potassium supplement therapy(8 mEq/day). 2) During the period of K supplement, the level of serum uric acid showed less elevation. 3) The side reaction of KCI continus tablet were mild indigestion which developed in two patients and disappeared soon without any particular treatment. In conclusion, the KCI continus tablet is useful in the prevention of hypopotassemia during chronic diuretic therapy.

Clinical Cardiac Electrophysiological Study on the Sinus Node and Atrioventricular Conduction System

Clinical EPS was performed in 16 normal adults without evidence of conduction disease on the surface standard 12 lead electrocardiogram in order to provide normal electrophysiological values of the sinus node function and AV conduction. EPS was also performed in 15 patients with sick sinus syndrome and 10 patients with AV conduction disturbance to evaluate the clinical usefulness of EPS in detecting sinus node dysfunction and AV conduction disturbance. The results were as follows. 1) The results of sinus node function test in the normal group were m-SNRT 853+/-198msec(range 800-1,560msec), c-SNRT 230+/-66msec(range 120-370msec), and %m -SNRT/SCL 127+/-11%(range 114-149%). 2) In 15 patients with SSS, the M-SNRT were ranged from 1,270 to 12,330msec and 10 patients(66%) had significantly increased m-SNRT exceeding 1,560msec. The c-SNRT were ranged from 230 to 10,730msec and 13 patients(83%) had significantly increased c-SNRT exceeding 370msec. The % m-SNRT/SCL were ranged from 136 to 770% and 12 patients(80%) had significantly increased % m-SNRT/SCL exceeding 150%. 3) The SACT in normal group were 84+/-14msec(range 70-105msec) measured by continuous atrial pacing method and 80+/-19 msec(range 60-115msec) measured by atrial extrastimulation method. 4) In SSS, the SACT measured by continuous atrial pacing method was ranged from 80 to 1,050msec and 11/12 patients(92%) had significantly increased SACT exceeding 112 msec. The SACT measured by atrial extrastimulation method was ranged from 90 to 310msec and 7/8 patients(88%) had significantly increased SACT exceeding 118 msec. 5) C-SNRT, % m-SNRT/SCL, and SACT were more useful in detecting sinus node dysfunction than m-SNRT. 6) The AV conduction intervals in normal group were PA interval 17+/-6(range 5-25msec), AH interval 96+/-18 msec(range 70-135msec), and HV interval 46+/-7msec(range 35-55msec). 7) Rapid atrial pacing induced Wenckebach type second degree AV block proximal to H at pacing rate of 90 to 190/min in 14/16 normal adults. 2 patients maintained intact AV conduction upto maximum pacing rate of 200/min. 8) His bundle electrogram showed the site of AV block in 9 of 10 patients with AV conduction disturbances. The sites of AV block were AV nodal area 1 case, intraHis bundle 4 cases, and infraHis bundle 4 cases. 9) EPS provided a good supportive information that was useful in selecting pacemaker therapy in a patient with chronic bifascicular block who revealed prolonged HV interval and infraHis bundle block at a pacing rate of 70min. 10) The refractory periods of AV conduction system in normal group were AERP 274+/-54msec (range 170-410msec), AVN-FRp 467+/-74msec(range 285-600msec), AVN-ERP 341+76msec(range 190-460), and V-ERP 280+/-25msec(range 240-320msec).