Development of controlled drug release systems based on thiolated polymers.

The purpose of the present study was to generate mucoadhesive matrix-tablets based on thiolated polymers. Mediated by a carbodiimide, L-cysteine was thereby covalently linked to polycarbophil (PCP) and sodium carboxymethylcellulose (CMC). The resulting thiolated polymers displayed 100+/-8 and 1280+/-84 micromol thiol groups per gram, respectively (means+/-S.D.; n=6-8). In aqueous solutions these modified polymers were capable of forming inter- and/or intramolecular disulfide bonds. The velocity of this process augmented with increase of the polymer- and decrease of the proton-concentration. The oxidation proceeded more rapidly within thiolated PCP than within thiolated CMC. Due to the formation of disulfide bonds within thiol-containing polymers, the stability of matrix-tablets based on such polymers could be strongly improved. Whereas tablets based on the corresponding unmodified polymer disintegrated within 2 h, the swollen carrier matrix of thiolated CMC and PCP remained stable for 6.2 h (mean, n=4) and more than 48 h, respectively. Release studies of the model drug rifampicin demonstrated that a controlled release can be provided by thiolated polymer tablets. The combination of high stability, controlled drug release and mucoadhesive properties renders matrix-tablets based on thiolated polymers useful as novel drug delivery systems.     

Oral insulin delivery: the potential of thiolated chitosan-insulin tablets on non-diabetic rats.

It was the aim of this study to develop a delivery system providing an improved efficacy of orally administered insulin utilizing a thiolated polymer. 2-Iminothiolane was covalently linked to chitosan. The resulting chitosan-TBA (chitosan-4-thiobutylamidine) conjugate exhibited 453.5+/-64.1 micromol thiol groups per gram polymer. 3.1% of these thiol groups were oxidised. Additionally, the enzyme inhibitors BBI (Bowman-Birk-Inhibitor) and elastatinal were covalently linked to chitosan representing 3.5+/-0.1% and 0.5+/-0.03% of the total weight of the resulting polymer conjugate, respectively. Chitosan-TBA conjugate (5 mg), insulin (2.75 mg), the permeation mediator reduced glutathione (0.75 mg) and the two inhibitor conjugates (in each case 0.75 mg) were compressed to so-called chitosan-TBA-insulin tablets. Control tablets consisted of unmodified chitosan (7.25 mg) and insulin (2.75 mg). Chitosan-TBA-insulin tablets showed a controlled release of insulin over 8 h. In vitro mucoadhesion studies showed that the mucoadhesive/cohesive properties of chitosan were at least 60-fold improved by the immobilisation of thiol groups on the polymer. After oral administration of chitosan-TBA-insulin tablets to non-diabetic conscious rats, the blood glucose level decreased significantly for 24 h corresponding to a pharmacological efficacy of 1.69+/-0.42% (means+/-S.D.; n=6) versus s.c. injection. In contrast, neither control tablets nor insulin given in solution showed a comparable effect. According to these results the combination of chitosan-TBA, chitosan-enzyme-inhibitor conjugates and reduced glutathione seems to represent a promising strategy for the oral application of insulin.     

Development and in vitro evaluation of a mucoadhesive vaginal delivery system for progesterone.

The purpose of the present study was to design a novel carrier system based on a mucoadhesive polymer exhibiting improved properties concerning drug delivery to the vaginal mucosa. This was reached by the covalent attachment of L-cysteine to commercially available polyacrylic acid (Carbopol 974P). Mediated by a carbodiimide, increasing amounts of L-cysteine were covalently linked to the polymer. The resulting thiolated polyacrylic acid conjugates (NaC974P-Cys) displayed between 24.8 and 45.8 micromol thiol groups per gram of polymer. Because of the formation of intra- and/or intermolecular disulfide bonds, the viscosity of an aqueous thiolated polymer gel (3%) increased about 50% at pH 7.0 within 1 h. In oscillatory rheological measurements, it was shown that this increase in viscosity is mainly due to the increase in elasticity. Tensile studies carried out on freshly excised cow vagina demonstrated a significant (P<0.05) increase in the total work of adhesion (TWA) compared to the unmodified polymer. An amount of 24.8 micromol thiol groups per gram of polymer resulted in a 1.45-fold increase in the TWA, whereas an amount of 45.8 micromol showed an even 2.28-fold increase. These improved mucoadhesive properties can be explained by the formation of disulfide bonds between the thiolated polymer and cysteine rich subdomaines of the mucus layer. The release rate of the model drug progesterone from tablets based on microcrystalline cellulose serving as the reference was approximately 1% per hour, whereas it was 0.58% per hour for the unmodified polymer (NaC974P) and 0.12% per hour for the thiolated polymer (NaC974P-Cys). Therefore, this thiolated polymer is a promising carrier for progesterone providing a prolonged residence time and a controlled drug release.     

Development and in vivo characterization of a novel peptide drug delivery system providing extended plasma half life

It was the aim of this study to develop a sustained parenteral peptide (DALCE) delivery system by the immobilization of DALCE to thiolated carboxymethyl dextran-cysteine (CMD-Cys) via disulfide bond formation. The resulting CMD-Cys-DALCE conjugate displayed a 22.6±7.9% (m/m) of DALCE (mean±S.D.; n=3). The conjugation of DALCE with CMD-Cys was confirmed by FTIR-ATR spectroscopy. In vitro release studies of conjugate CMD-Cys-DALCE in the presence of 2 μM/ml reduced glutathione (GSH) being also available in the plasma showed a sustained peptide release over a time period of 8 h, because of thiol/disulfide exchange reactions. For in vivo pharmacokinetic study, DALCE and CMD-Cys-DALCE were administered intravenously to male Sprague-Dawley rats at a dose of 1mg/kg. The AUC(0-8) (ng.min/ml) was determined to be 268848±924 and 40019±495 for CMD-Cys-DALCE and DALCE, respectively. The mean residence time (MRT) was determined to be 256±8 and 53.1±9.5 min for CMD-Cys-DALCE and for DALCE, respectively. CMD-Cys-DALCE showed a more than 5-fold increased elimination half-life (p<0.01), 3-fold decreased volume of distribution (p<0.01) and a 6.7-fold decreased plasma clearance rate (p<0.01) compared to DALCE. According to these findings, CMD-Cys-DALCE seems to act as prodrug by improving half-life and decreasing plasma clearance.     

Systemic peptide delivery via the stomach: in vivo evaluation of an oral dosage form for salmon calcitonin.

It was the aim of this study to investigate the potential of stomach targeted delivery systems for systemic peptide administration using salmon calcitonin as a model drug. Chitosan was modified by the immobilization of thiol groups utilizing 2-iminothiolane in order to obtain a chitosan-4-thiobutylamidine conjugate (chitosan-TBA). Furthermore, a chitosan-pepstatin A conjugate was synthesized by a carbodiimide mediated linkage of the pepsin inhibitor to the polymer. The protective effect of this novel conjugate for calcitonin towards pepsin was evaluated in vitro. Minitablets (5 mg) were generated by direct compression of calcitonin, chitosan, chitosan-TBA, chitosan-pepstatin A conjugate and glutathione (GSH), respectively (A, 1:0:69:20:10; B, 1:79:0:20:0; C, 1:99:0:0:0). The drug release was investigated in an artificial gastric fluid. Biofeedback studies were performed in rats by determining the decrease in plasma calcium level after oral administration. The novel chitosan-pepstatin A conjugate displayed 291+/-58 nmol inhibitor per gram polymer (mean+/-S.D., n = 5). The chitosan-inhibitor conjugate showed a very strong protective effect for salmon calcitonin towards pepsinic degradation. A controlled drug release was provided by all tested dosage forms-A, B and C. Dosage form B led only to a slight reduction of the plasma calcium level, displaying a pharmacological efficacy versus i.v. injection of 0.41%, while dosage form C did not lead to any significant effect. In contrast, dosage form A led to a decrease in the plasma calcium level of 10% for at least 12 h. The pharmacological efficacy of this formulation was determined to be 1.35%. The study suggests that stomach targeted oral delivery might be a promising novel approach for noninvasive systemic peptide administration.     

Incidence of T Wave Alternation After Acute Myocardial Infarction and Correlation with Other Prognostic Parameters: Results of a Prospective Study

Tachycardia induced alternation of the T wave (TWA) has been associated with arrhythmia morbidity in mixed patient populations. However, less is known concerning the general incidence of TWA and its usefulness in risk stratification early after acute myocardial infarction (MI). TWA was prospectively and systematically assessed in 140 consecutive patients 15 +/- 6 days after acute MI and prior to discharge. Results of TWA measurements were compared to other noninvasive risk markers, LV function, and coronary angiography. Sustained TWA was present at rest or inducible during exercise in 27% of patients. The patient-specific heart rate for the onset of TWA was 98 +/- 9 beats/min. After multivariate analysis, TWA correlated with age (P = 0.02) and LV function (P = 0.002) and occurred more often in patients after nonanterior MI (P = 0.03). Acute results of Holter monitoring, late potentials by signal-averaged ECG, and heart rate variability were unrelated to the TWA status. During follow-up (451 +/- 210 days) two major arrhythmic events occurred. The incidence of TWA early after MI is about 25%. TWA is related to age and LV function but not to other common arrhythmia markers. Although TWA does not appear to be related to excessive cardiac morbidity, evaluation of the prognostic significance of TWA requires further study.     

Plasma-volume contraction and exercise-induced hypoxaemia modulate erythropoietin production in healthy humans

This study examined exercise-induced hypoxaemia (EIH) and plasma volume contraction as modulators of serum erythropoietin (Epo) production. Five athletes cycled for 3 min at supra-maximal power outputs, at each of two different elevations (1,000 m and 2,100 m). Five subjects were exposed to normobaric hypoxia (F(I)O(2)=0.159), seven subjects underwent plasmapheresis to reduce plasma volume and eight subjects were time controls for Epo levels. Oxyhaemoglobin saturation was significantly reduced during exercise and during normobaric hypoxia. The time period of haemoglobin oxygen saturation <91% was 24+/-29 s (mean+/-S.D., n=5) for exercise at 1000 m, 136+/-77 s (mean+/-S.D., n=5) for exercise at 2100 m and 178+/-255 s (mean+/-S.D., n=5) with resting hypoxic exposure. However, significantly increased serum Epo levels were observed only following exercise (24+/-3%; mean+/-S.D., n=5 at 1,000 m and 36+/-5%; mean+/-S.D., n=5 at 2,100 m). Volume contraction also resulted in increased serum Epo (35+/-6%; mean+/-S.D., n=7) in spite of a significant rise in haematocrit of 2.2%. Despite similar degrees of arterial desaturation, only the hypoxaemia induced by exercise was associated with an increase in serum Epo. This finding indicates that other factors, in addition to hypoxaemia, are important in modulating the production of Epo in response to exercise. Volume depletion in the absence of exercise resulted in increases in Epo levels that were comparable with those observed in response to exercise. The paradoxical responses of the increased haematocrit and the increase in Epo in subjects undergoing plasmapheresis suggests that plasma volume may also modulate the production of Epo.     

Cerebral oxygenation at high altitude and the response to carbon dioxide, hyperventilation and oxygen. The Birmingham Medical Research Expeditionary Society

Cerebral oxygenation is likely to be of critical importance in determining function at high altitude. The present study has used the technique of near-IR spectroscopy to monitor changes in cerebral regional oxygenation in response to inhaled carbon dioxide, hyperventilation and supplementary oxygen on ascent to 4680 m over 3 days. At sea level, inhaled CO(2) resulted in a significant rise in cerebral regional oxygenation [from mean 69.6% (S.D. 2.4% to 71. 1+/-2.3%; means+/-S.D.; P<0.001). At 4680 m, CO(2) increased regional cerebral oxygenation (63.8+/-2.5% to 65.9+/-2.2%; P<0.001) and also increased peripheral oxygen saturation (75.1+/-6.1% to 83. 6+/-4.0%; P<0.001). Voluntary hyperventilation resulted in improved peripheral oxygen saturation at 2770 m, 3650 m and 4680 m, whereas cerebral regional oxygenation was reduced at sea level and at 2770 m, unchanged at 3650 m and increased at 4680 m. Supplementary oxygen (6 1itres/min) at 4680 m resulted in greater improvements in peripheral oxygen saturation (76.7+/-7.9% to 98.1+/-1.5%; P<0.001) and cerebral regional oxygenation (64.6+/-3.3% to 70.6+/-2.9%; P<0. 001) than were found with CO(2) or hyperventilation. We conclude that attempts to increase CO(2) inhalation or ventilation at high altitude are likely to be beneficial for cerebral oxygenation in the short term.     

T wave alternans as a risk predictor in patients with cardiomyopathy and mild-to-moderate heart failure

The analysis of t wave alternans (TWA) was introduced to identify patients with an increased risk of ventricular tachyarrhythmias. The inducibility of ventricular tachyarrhythmias and the spontaneous arrhythmic events are correlated with a positive TWA in patients with a reduced left ventricular ejection fraction and survived myocardial infarction. In contrast, this study is the first to investigate the correlation of a survived sudden cardiac death and TWA in patients without coronary heart disease and only slightly decreased left ventricular function. Sixty patients were included in the study. The TWA analysis was performed using the Cambridge Heart system (CH2000). Patients were sitting on a bicycle ergometer and exercised with a gradual increase of workload to maintain a heart rate of at least 105 beats/min. The exercise test was stopped after recording 254 consecutive low noise level heart beats. The electrocardiographic signals were digitally processed using a spectral analysis method. The magnitude of TWA was measured at a frequency of 0.5 cycles/beat. A TWA was defined as positive if the ratio between TWA and noise level was > 3.0 and the amplitude of the TWA was > 1.8 microV. Twelve (20%) of the included 60 patients showed a positive TWA. The sensitivity concerning a previous arrhythmic event amounted to 65%, the specificity up to 98%, respectively. The alternans ratio was significantly higher in patients with a previous event (30.3 +/- 53.2 vs 2.9 +/- 5.9, P < 0.001) and cumulative alternans voltage (4.67 +/- 3.55 vs 1.75 +/- 1.88 microV, P < 0.001). In 19 patients, invasively investigated by an electrophysiological study, a significant correlation between inducibility of tachyarrhythmias and a positive TWA result was found (Spearman R = 0.51, P = 0.01). In conclusion, the TWA analysis seems to identify patients with nonischemic cardiomyopathy who are at an increased risk of ventricular tachyarrhythmias.     

Heterologous enzyme immunoassay for serum androstenediol

A heterologous enzyme immunoassay for serum androstenediol (Adiol: 3beta, 17beta-dihydroxy-androst-5-ene) was established. The combination of anti-Adiol antiserum raised in rabbit against Adiol 7-O-(carboxymethyl)oxime (Adiol 7-CMO) conjugated bovine serum albumin (Adiol 7-CMO-BSA) and Adiol 7-iminomethylcarboxylic acid conjugated alkaline phosphatase was used for the assay. The sensitivity of the heterologous assay system was superior to that of a homologous assay system in which an antibody raised in rabbit against Adiol 7-CMO-BSA and enzyme labeled antigen, Adiol 7-CMO conjugated alkaline phosphatase, were used. The minimal amount of Adiol detected was 0.4 ngml(-1) and the measurable range was from 0. 4 to 150 ngml(-1). Intra-assay coefficients of variation (C.V.) were 8.6% (1.52+/-0.13 ngml(-1), mean+/-S.D., n=10) and 6.7% (13.4+/-0.9 ngml(-1), n=10). Inter-assay C.V. were 12.9% (1.63+/-0.21 ngml(-1), n=8) and 11.5% (12.2+/-1.4 ngml(-1), n=8). A linear relation was observed between the serum sample dilution and the Adiol concentration. For recovery study, authentic Adiol was added to serum sample (original concentration: 1.43 ngml(-1)). The calculated final Adiol concentration was 2.99 ngml(-1). The recovery was 98.6% (n=5). The Adiol concentrations in healthy subjects measured by the proposed assay (male: 1.1+/-0.3 ngml(-1) (mean+/-S.D.), range: 0.7-1. 7 ngml(-1), age: 22-50, n=10; female: 0.6+/-0.4 ngml(-1), range: 0. 2-1.6 ngml(-1), age: 23-48, n=20) were consistent with reported values.     

Vitamin D3 uptake by the isolated perfused rat liver from lipoprotein fractions is separate from cholesterol and triglyceride uptake

Using the isolated perfused rat liver we examined the uptake of [14C] or [3H] vitamin D3 and [14C] triglycerides or [3H] cholesterol by the liver of normal rats, from different lipoprotein fractions, as measured by disappearance from the perfusate. When incorporated into chylomicrons only 43% of the vitamin D3 remained in the perfusate at 60 min (i.e. 57% uptake) as compared to 68% of the triglycerides (i.e. 32% uptake). When added on very low density lipoproteins (VLDL) at 60 min 37 +/- 3% (n = 6) of the vitamin D3, 38 +/- 2% of the cholesterol (n = 3) (P NS), and 83 +/- 4% of the triglycerides (n = 3) remained in the perfusate (P less than 0.0005 for cholesterol:triglycerides and vitamin D3:triglycerides). For high density lipoprotein fraction (HDL) isolated perfused livers were studied with and without albumin present in the perfusate. Without albumin 19 +/- 8% (n = 3) of the vitamin D3 and 43 +/- 8% (n = 3) of the cholesterol remained in the perfusate at 60 min. The results with albumin present were 40 +/- 1% (n = 5) of the vitamin D3 and 63 +/- 4% (n = 5) of the cholesterol at 60 min (P less than 0.0005). The cholesterol:cholesterol ester ratio of the VLDL fraction was 8.8:1 and of the HDL fraction 1:1.4. There was no metabolism of vitamin D3 during the 1 h perfusion. These results suggest that vitamin D3 is in equilibrium with the lipoprotein surface, and the hepatic uptake of vitamin D is a surface phenomenon independent of lipoprotein metabolism.     

The influence of beta-adrenergic agonists and antagonists on T-wave alternans in patients with and without ventricular tachyarrhythmia

BACKGROUND: T-wave alternans (TWA) is an important noninvasive measurement of ventricular tachyarrhythmia (VT) and is known to be influenced by the sympathetic nervous system. We examined the correlation between TWA measurement and the sympathetic nervous system in patients with and without VT. METHODS AND RESULTS: Thirty-five patients (28 men, 7 women; mean age, 59 +/- 15 years) with tachyarrhythmia were assigned to two groups: VT group (n = 15) and supraventricular tachyarrhythmia (SVT) group (n = 20). Alternans voltage in lead vector magnitude (eVM) was measured during atrial pacing (90, 110 beats/min (bpm)). After eVM was measured at baseline, propranolol was administered, and eVM was measured again. In a subset of 18 patients (10 with VT and 8 with SVT), isoproterenol was administered prior to propranolol infusion. After propranolol infusion, eVM of both the VT and the SVT groups decreased significantly compared to baseline. The changes in absolute value of eVM at 110 bpm after propranolol infusion were greater in the VT group than in the SVT group (-1.3 +/- 0.8 microV vs -0.5 +/- 0.8 microV, P < 0.05). The eVM values of both the VT and the SVT groups increased after administration of isoproterenol compared to the baseline value. The changes in absolute value and percentile of eVM after isoproterenol infusion were smaller in the VT group than in the SVT group (2.0 +/- 1.8 microV vs 3.9 +/- 3.5 microV, P < 0.05; 21 +/- 18% vs 48 +/- 36%, P < 0.05). CONCLUSIONS: The sympathetic nervous system has an influence over microvolt-level TWA. Administration of a beta-adrenergic antagonist caused a significant decrease in TWA, particularly in the VT group. This may partially explain the mechanism by which adrenergic antagonists inhibit VTs.     

The effect of different buffers and amounts of intestinal alkaline phosphatase isoforms on total alkaline phosphatase activity

BACKGROUND: The transphosphorylating accepter buffers (2-amino-2-methyl-1-propanol, AMP; N-methyl-D-glucamine, MEG; diethanolamine, DEA and 2-ethylaminoethanol, EAE) have been widely used for the measurement of serum total alkaline phosphatase activity (ALP) in clinical laboratories, and the individual isozyme are activated differently by respective buffers. MATERIALS AND METHODS: We examined the activity of serum ALP using four buffers with levels of both high molecular weight intestinal alkaline phosphatase (HIAP) and normal molecular weight intestinal alkaline phosphatase (NIAP). We classified 80 healthy subjects into two groups of blood group B or O secretors (n=36) and other blood groups (n=44). RESULTS: The mean ALP activities at fasting in blood group B or O secretors from AMP, MEG, DEA and EAE methods were 15.5%, 24.0%, 11.0% and 22.1% higher than those in other blood groups, respectively. The reference ranges of ALP activity at fasting with the AMP method in blood group B or O secretors and other blood groups were 63.5+/-17.4 U/l (mean+/-S.D.) and 55.0+/-14.5 U/l (mean+/-S.D.), respectively. The difference between the reference ranges of ALP activity in blood group B or O secretors and other blood groups was statistically significant (p<0.01). HIAP and NIAP in serum at fasting only appeared in blood group B or O secretors, and the activities of HIAP and NIAP were 4.7+/-3.4 U/l (mean+/-S.D.) and 2.2+/-1.2 U/l (mean+/-S.D.), respectively. The activity of ALP-(HIAP+NIAP) in blood group B or O secretors was 56.6+/-15.1 U/l (mean+/-S.D.), and this reference range was approximately the same as the ALP activity (55.0+/-14.5 U/l) of other blood groups. The same results were observed with MEG, DEA and EAE methods. CONCLUSIONS: These results suggested that the differences in ALP activity in blood group B or O secretors and other blood groups were closely related to the HIAP and NIAP levels.     

Relationship between abnormal microvolt T-wave alternans and poor glycemic control in type 2 diabetic patients

BACKGROUND: Abnormal microvolt T-wave alternans (TWA) predicts the risk of ventricular arrhythmias and sudden cardiac death. Although type 2 diabetes is associated with an increased risk of these events, there is a dearth of available data on microvolt TWA measurements in type 2 diabetic populations. METHODS: We studied 59 consecutive type 2 diabetic outpatients without manifest cardiovascular disease (CVD) and 35 non-diabetic controls who were matched for age, sex, and blood pressure values. Microvolt TWA analysis was performed non-invasively using the CH-2000 system during a sub-maximal exercise with the patient sitting on a bicycle ergometer. RESULTS: The frequency of abnormal TWA was significantly higher in diabetic patients than in controls (25.4 vs 5.7%; P < 0.01). Among diabetic patients, those with abnormal TWA (n = 15) had remarkably higher hemoglobin A1c (HbA1c) (8.1 +/- 0.9 vs 7.1 +/- 0.8%, P < 0.001) and slightly smaller time-domain heart rate variability parameters (i.e., RMSSD, root mean square of difference of successive R-R intervals) than those with normal TWA (n = 44). Gender, age, body mass index, lipids, blood pressure values, cigarette smoking, diabetes duration, microvascular complication status, QTc interval, and current use of medications did not significantly differ between the groups. In multivariate regression logistic analysis, HbA1c (OR 13.6, 95% CI 2.0-89.1; P = 0.0076) predicted abnormal TWA independent of RMSSD values and other potential confounders. CONCLUSIONS: Our findings suggest that abnormal TWA is a very common condition (approximately 25%) among people with type 2 diabetes without manifest CVD and is closely correlated to glycemic control.     

Predicting arrhythmia-free survival using spectral and modified-moving average analyses of T-wave alternans

BACKGROUND: T-wave alternans (TWA) is a promising electrocardiogram (ECG) predictor of sudden cardiac arrest, yet needs specialized recordings for conventional spectral analysis. Modified moving average (MMA) analysis is a new approach that can measure TWA from routine ECGs, thus widening its applicability. However, MMA-TWA has not been calibrated against spectral TWA nor outcome in high risk patients. We hypothesized that spectral and MMA-TWA would both predict arrhythmia-free survival on long-term prospective follow-up. METHODS AND RESULTS: In 41 patients with left ventricular systolic dysfunction (ejection fraction 31 +/- 13%), we studied TWA simultaneously using spectral and MMA during pacing (< 110 beats/min). MMA amplified TWA over spectral analyses (13.0 +/- 8.28 microV vs 1.96 +/- 5.15 microV, P < 0.001). On 542 +/- 311 days' follow-up, from clinic visits, telephonic interviews, and device interrogations, there were 11 deaths or sustained ventricular arrhythmias ('events'). Positive spectral TWA (>or=1.9 microV) identified patients with from those without events (P = 0.02). Receiver-operating characteristics for MMA-TWA showed that the cutpoint >or= 10.75 microV was optimal for the combined endpoint. Kaplan-Meier analysis using this MMA-TWA cutpoint trended to predict events (P = 0.06), while MMA combined with spectral TWA identified events (P = 0.01). CONCLUSIONS: MMA amplifies TWA compared to traditional spectral analyses, but both likely reflect similar pathophysiology. Validation in larger populations will enable MMA-TWA to be widely applied to stratify risk for sudden cardiac arrest.     

The use of thiolated polymers as carrier matrix in oral peptide delivery--proof of concept.

It was the aim of this study to develop an oral delivery system for the peptide drug antide. The stability of the therapeutic peptide towards gastrointestinal peptidases was evaluated. The therapeutic agent and the permeation mediator glutathione were embedded in the thiolated polymer chitosan-4-thio-butylamidine conjugate (chitosan-TBA conjugate) and compressed to tablets. Drug release studies were performed in the dissolution test apparatus according to the Pharmacopoeia Europea using the paddle method and demineralized water as release medium. In order to avoid mucoadhesion of these delivery systems already in the oral cavity and oesophagus tablets were coated with a triglyceride. These tablets were orally given to pigs (weight: 50+/-2 kg; Edelschwein Pietrain). Moreover, antide was administered intravenously, subcutaneously and orally in solution. Results showed stability of antide towards pepsin, trypsin and chymotrypsin. In contrast, antide was rapidly degraded by elastase. Consequently a stomach-targeted delivery system was designed. Drug release studies demonstrated an almost zero-order controlled release of antide over 8 h. In vivo studies demonstrated a relative bioavailability of 34.4% for the subcutaneous administration. Oral administration of antide in solution led to no detectable concentrations of the drug in plasma at all. In contrast, administering antide being incorporated in the thiolated polymer resulted in a significant uptake of the peptide. The absolute and relative bioavailability was determined to be 1.1% and 3.2%, respectively.     

Differences in lymph drainage between swollen and non-swollen regions in arms with breast-cancer-related lymphoedema

Recent research indicates that the pathophysiology of breast-cancer-related lymphoedema (BCRL) is more complex than simple axillary lymphatic obstruction as a result of the cancer treatment. Uneven distribution of swelling (involvement of the mid-arm region is common, but the hand is often spared) is puzzling. Our aim was to test the hypothesis that local differences in lymphatic drainage contribute to the regionality of the oedema. Using lymphoscintigraphy, we measured the removal rate constant, k (representing local lymph flow per unit distribution volume, VD), for 99mTc-labelled human immunoglobulin G in the oedematous proximal forearm, and in the hand (finger web) in women in whom the hand was unaffected. Tracer was injected subcutaneously, and the depot plus the rest of the arm was monitored with a gamma-radiation camera for up to 6 h. VD was assessed from image width. Contralateral arms served as controls. k was 25% lower in oedematous forearm tissue than in the control arm (BCRL, -0.070+/-0.026% x min(-1); control, -0.093+/-0.028% x min(-1); mean+/-S.D.; P=0.012) and VD was greater. In the non-oedematous hand of the BCRL arm, k was 18% higher than in the control hand (BCRL, -0.110+/-0.027% x min(-1); control, -0.095+/-0.028% x min(-1); P=0.057) and 59% higher than forearm k on the BCRL side (P=0.0014). VD did not differ between the hands. Images of the BCRL arm following hand injection showed diffuse activity in the superficial tissues, sometimes extending almost to the shoulder. A possible interpretation is that the hand is spared in some patients because local lymph flow is increased and diverted along collateral dermal routes. The results support the hypothesis that regional differences in surviving lymphatic function contribute to the distribution of swelling.     

Studies on coproporphyrin isomers in urine and feces in the porphyrias.

The urinary and fecal distribution and the relative proportions of the four coproporphyrin (copro) isomers I-IV were analysed in 20 healthy subjects and in patients suffering from one of the seven common types of hepatic or erythropoietic hereditary porphyrias. The ratios of copro isomers I-IV were analyzed by ion-pair high-performance liquid chromatography (HPLC). Observations showed significantly increased proportions of fecal copro isomer I and decreased proportions of copro isomers III, II and IV in erythropoietic porphyrias. In acute hepatic porphyrias the excretion of fecal copro isomer III is dominant (isomer III = 58.4+/-24.0%; x+/-S.D.) and significantly higher (P < 0.001) than in erythropoietic porphyrias (isomer III = 15.3+/-7.7%; x+/-S.D.) and chronic hepatic porphyrias (isomer III = 25.8+/-7.6%; x+/-S.D.). The increased proportions of fecal copro isomer III proved to be important for the diagnosis of hereditary coproporphyria and porphyria variegata independent of the clinical phase existing. These last two acute hepatic porphyrias also showed markedly elevated percentages of the fecal atypical isomers II and IV. In urine significantly decreased proportions of copro isomer I in acute hepatic porphyrias (isomer I = 12.3+/-6.0%; x+/-S.D.) could be observed as compared with non-acute porphyrias (isomer I = 53.7+/-15.2%; x+/-S.D.). Conversely, the proportion of urinary copro isomer III was significantly higher in acute hepatic porphyrias (isomer III= 81.4+/-6.4%; x+/-S.D.). As expected, the greatest amounts of urinary copro isomer I were found in congenital erythropoietic porphyria (isomer I =92.0+/-3.3; x+/-S.D.) and protoporphyria with hepatobiliary complications (isomer I = 81.3+/-10.7; x+/-S.D.). The atypical urinary copro isomers I1 and IV were detected in all types of porphyrias ranging from 0.1 to 11.5%. The combined amounts of copro isomers II and IV show a significantly decreased percentage in congenital erythropoietic porphyria as compared with all other types of hereditary porphyrias. In conclusion, we demonstrate that the characteristic pattern of the copro isomer constellations I-IV in the various types of porphyrias are of differential diagnostic importance. The inversion of the I to III ratio in feces in hereditary coproporphyria and porphyria variegata allows the recognition of gene carriers.     

Abnormal heart rate turbulence predicts the initiation of ventricular arrhythmias

BACKGROUND: Abnormal heart rate turbulence (HRT) reflects autonomic derangements predicting all-cause mortality, yet has not been shown to predict ventricular arrhythmias in at-risk patients. We hypothesized that HRT at programmed ventricular stimulation (PVS) would predict arrhythmia initiation in patients with left ventricular dysfunction. METHODS: We studied 27 patients with coronary disease, left ventricular ejection fraction (LVEF) 26.7 +/- 9.1%, and plasma B-type natriuretic peptide (BNP) 461 +/- 561 pg/mL. Prior to arrhythmia induction at PVS, we measured sinus cycles after spontaneous or paced premature ventricular contractions (PVCs) for turbulence onset (TO; % cycle length change following PVC) and slope (TS; greatest slope of return to baseline cycle). T-wave alternans (TWA) was also measured during atrial pacing. RESULTS: At PVS, abnormal TO (> or =0%) predicted inducible ventricular tachycardia (VT; n = 10 patients; P < 0.05). TO was greater in inducible than in noninducible patients (2.3 +/- 3.1% vs -0.02 +/- 2.8%, P < 0.05) and correlated with LVEF (P < 0.05) but not with BNP. TS did not differ between groups. Conversely, ambulatory HRT differed significantly from HRT at PVS (TO -0.55 +/- 1.08% vs 0.85 +/- 3.02%, P < 0.05; TS 2.63 +/- 2.09 ms/RR vs 8.70 +/- 6.56 ms/RR, P < 0.01), and did not predict inducible VT but trended (P = 0.05) to predict sustained VT on 739 +/- 179 days follow-up. TWA predicted inducible (P < 0.05) and spontaneous (P = 0.0001) VT but did not co-migrate with HRT. CONCLUSIONS: Abnormal HRT measured at PVS predicted the induction of sustained ventricular arrhythmias in patients with ischemic cardiomyopathy. However, HRT at PVS did not correlate with ambulatory HRT, nor with TWA, both of which predicted spontaneous ventricular arrhythmias. Thus, HRT may reflect the influence of autonomic milieu on arrhythmic susceptibility and is likely complementary to traditional arrhythmic indices.     

Regulation of myocardial amino acid balance in the conscious dog.

The effects in vivo of physiologic increases in insulin and amino acids on myocardial amino acid balance were evaluated in conscious dogs. Arterial and coronary sinus concentrations of amino acids and coronary blood flow were measured during a 30-min basal and a 100-min experimental period employing three protocols: euglycemic insulin clamp (plasma insulin equaled 70 +/- 11 microU/ml, n = 6); euglycemic insulin clamp during amino acid infusion (plasma insulin equaled 89 +/- 12 microU/ml, n = 6); and suppression of insulin with somatostatin during amino acid infusion (plasma insulin equaled 15 +/- 4 microU/ml, n = 6). Basally, only leucine and isoleucine were removed significantly by myocardium (net branched chain amino acid [BCAA] uptake equaled 0.5 +/- 0.2 mumol/min), while glycine, alanine, and glutamine were released. Glutamine demonstrated the highest net myocardial production (1.6 +/- 0.2 mumol/min). No net exchange was seen for valine, phenylalanine, tyrosine, cysteine, methionine, glutamate, asparagine, serine, threonine, taurine, and aspartate. In group I, hyperinsulinemia caused a decline of all plasma amino acids except alanine; alanine balance switched from release to an uptake of 0.6 +/- 0.4 mumol/min (P less than 0.05), while the myocardial balance of other amino acids was unchanged. In group II, amino acid concentrations rose, and were accompanied by a marked rise in myocardial BCAA uptake (0.4 +/- 0.1-2.6 +/- 0.3 mumol/min, P less than 0.001). Uptake of alanine was again stimulated (0.9 +/- 0.3 mumol/min, P less than 0.01), while glutamine production was unchanged (1.3 +/- 0.4 vs. 1.6 +/- 0.3 mumol/min). In group III, there was a 4-5-fold increase in the plasma concentration of the infused amino acids, accompanied by marked stimulation in uptake of only BCAA (6.8 +/- 0.7 mumol/min). Myocardial glutamine production was unchanged (1.9 +/- 0.4-1.3 +/- 0.7 mumol/min). Within the three experimental groups there were highly significant linear correlations between myocardial uptake and arterial concentration of leucine, isoleucine, valine, and total BCAA (r = 0.98, 0.98, 0.92, and 0.97, respectively); P less than 0.001 for each). In vivo, BCAA are the principal amino acids taken up by the myocardium basally and during amino acid infusion. Plasma BCAA concentration and not insulin determines the rate of myocardial BCAA uptake. Insulin stimulates myocardial alanine uptake. Neither insulin nor amino acid infusion alters myocardial glutamine release.