Injectable long-acting systems for Radix Ophiopogonis polysaccharide based on mono-PEGylation and in situ formation of a PLGA depot.

BACKGROUND: Radix Ophiopogonis polysaccharide (ROP), a highly hydrophilic macromolecule, has a unique anti-ischemic action in the myocardium. One of the main problems with its use is its relatively short half-life in vivo. To solve this problem, injectable long-acting drug delivery systems, which combine mono-PEGylation (PEG, polyethylene glycol) with the in situ formation of poly(D,L-lactide-co-glycolide) copolymer (PLGA) depots, were tested in this study. METHODS: Through a moderate coupling reaction between 20 kDa amino-terminated methoxy-PEG and excessive ROP with activated hydroxyls, a long-circulating and bioactive mono-PEGylated ROP was prepared and characterized. A reasonable and applicable range of PLGA formulations loaded with the mono-PEGylated ROP were prepared, characterized, and evaluated in vivo. RESULTS: Relative to ROP, the half-life of which was only 0.5 hours, the conjugate alone, following subcutaneous administration, showed markedly prolonged retention in the systemic circulation, with a mean residence time in vivo of approximately 2.76 days. In combination with in situ-forming PLGA depots, the residence time of the conjugate in vivo was prolonged further. In particular, a long-lasting and steady plasma exposure for nearly a month was achieved by the formulation comprising 40% 30 kDa PLGA in N-methyl-2-pyrrolidone. CONCLUSION: Long-lasting and steady drug exposure could be achieved using mono-PEGylation in combination with in situ formation of PLGA depots. Such a combination with ROP would be promising for long-term prophylaxis and/or treatment of myocardial ischemia. For high-dose and highly hydrophilic macromolecular drugs like ROP, more than one preparation technology might be needed to achieve week-long or month-long delivery per dosing.

Adaptation of an enzymatic polyfructosan assay to clinical practice.

Inulin or polyfructosan clearance is regarded as the most accurate method of assessing the glomerular filtration rate. We propose an enzymatic method of polyfructosan determination based on the hydrolysis of polyfructosan into fructose by inulinase and the elimination of the interfering quantity of glucose by glucose oxidase. This spectrophotometric microplate formatted assay, which demonstrated very good specificity and reproducibility (within-run precision <1% and between-run precision <3.5%), is cheap and simple to perform and can be used by all analytical laboratories and in all clinical conditions.

A fructan, from Radix ophiopogonis, stimulates the proliferation of cultured lymphocytes: structural and functional analyses.

A fructan, Opaw-2, with molecular mass of about 14 kDa, was isolated from the tuberous roots of Ophiopogon japonicus. Opaw-2 comprises fructose and glucose with a molar ratio of 30:1. Linkage and NMR analyses indicated that Opaw-2 has a backbone structure of beta-(1-->2)-Fruf and beta-(2-->6)-Fruf residues that branches at O-6 of beta-(1-->2)-Fruf residues with alpha-1-linkage to the Glcp residues and terminates with Fruf residues. In cultured lymphocytes, the application of Opaw-2 significantly stimulated the proliferation of lymphocytes in a dose-dependent manner. By using atomic force microscopy, Opaw-2 showed a morphological change from globular to helical fibrous shape at increasing concentrations.

Reducing shock number dramatically decreases lesion size in a juvenile kidney model.

BACKGROUND AND PURPOSE: Adult stone patients are treated with several thousand lithotripter shockwaves (SWs) in order to pulverize a kidney stone. This typical clinical dose assures that the stone will be fractured completely. However, this same dose induces damage to the kidney, especially pediatric-size kidneys. If increasing SW number is known to increase renal injury and functional impairment, will reducing SW number below typical treatment levels significantly decrease kidney damage and hemodynamic changes? MATERIALS AND METHODS: To address this question, one kidney in each of nine juvenile pigs (6-7 weeks old) was treated with 1000 SWs at 24 kV directed at a lower-pole calix with an unmodified HM-3 lithotripter. Parenchymal-lesion size was determined by sectioning the entire kidney and quantitating the amount of hemorrhage in each slice. Renal function was determined before and after SW treatment by inulin clearance, paraaminohippurate (PAH) extraction, and PAH clearance. The resulting morphologic and functional changes were then compared with those of kidneys that had been treated with a typical clinical dose of 2000 SWs (data previously published; J Am Soc Nephrol 2000;11:310). Eleven pigs were utilized as sham-treated controls. RESULTS: Limiting SW number to 1000 significantly reduced the size of the lesion (by 95%) and reduced the degree of functional change (glomerular filtration rate by 38%, PAH extraction by 73%, renal plasma flow by 46%) compared with kidneys receiving 2000 SWs (an adult dose). CONCLUSIONS: These data support the idea that SW number should be reduced to the lowest number that fractures kidney stones in order to minimize renal injury and functional impairment.

Determination of Ophiopogon japonicus polysaccharide in plasma by HPLC with modified postcolumn fluorescence derivatization.

Interest in antimyocardial ischemic activity of a graminan-type fructosan with an average molecular weight of 5,000 Da from Ophiopogon japonicus (FOJ-5) has necessitated the development of a sensitive and specific method to study its pharmacokinetics. An HPLC method with modified postcolumn fluorescence derivatization to determine FOJ-5 in plasma was developed in this study. The Shodex Sugar KS-802 high-performance gel column was chosen for separating FOJ-5 from its degradation products and endogenous carbohydrates. The postcolumn procedure involved acid hydrolysis of the column eluate at 150 degrees C, which decreased the detection limit for FOJ-5 from 1 microg to 25 ng, followed by fluorometric reaction with guanidine in an alkaline medium at 90 degrees C. The clearance of FOJ-5 from the bodies of rats following intravenous injection displayed a complex type of kinetics involving at least two compartments, and the half-life of the elimination of FOJ-5 from plasma administered at 15 mg/kg (18.1 min) was quicker than that administered at 50mg/kg (28.9 min). This modified approach can also be used for microanalysis of both nonreducing oligosaccharides and other neutral polysaccharides.

Plasma disappearance and distribution volume of polyfructosan in type 1 (insulin-dependent) diabetes mellitus.

The simultaneous plasma disappearance curves of the extracellular marker polyfructosan and 125I-fibrinogen were recorded for 3 h in 24 male long-term diabetic patients. Eight normal subjects served as a control group. The patients were divided into three groups according to their urinary albumin excretion: Group 1 had normal albumin excretion (< 30 mg/24 h), Group 2 had microalbuminuria (30-300 mg/24 h), and Group 3 had clinical nephropathy (> 300 mg/24 h). Using fibrinogen as a plasma volume reference, the permeability surface product (PdS), the plasma clearance rate at 10 min C1(10), and the extracellular volume (ECV) were determined from the polyfructosan curves. Plasma volume was similar in all groups, and no differences were found between Group 1 and the control group. PdS was higher in the albuminuric groups, but barely to reach significant levels (p = 0.08-0.03), while C1(10) was significantly elevated (p < 0.01). The ECV was also significantly higher in these patients (p = 0.02-0.005), resulting in a marked decrease of the plasma volume to extravascular volume ratio. The results suggest a general degeneration of perivascular resistance components in diabetic microvascular complications, followed by secondary alterations of fluid balance, due to decreasing colloid osmotic counterpressure and eventually increasing transcapillary hydrostatic pressure.

[Polyfructosan and para-aminohippuric acid clearance, comparison of 2 methods for determining glomerular filtration and plasma renal flow]. / Clearance del polifruttosano e dell'acido para-aminoippurico, confronto di due metodiche per la determinazione della filtrazione glomerulare e della portata renale plasmatica.

Glomerular Filtration Rate (GFR) and Renal Plasma Flow (RPF) were measured respectively, Polyfructosan and Para-aminohippuric acid clearances, in 13 patients with different degrees of renal insufficiency. Two different methods were used, the standard method and one without urine collection. The mean GFR (66.4 +/- 27.8 vs. 72.2 +/- 19.0 ml/m, r = 0.74) and RPF (431 +/- 204 vs 490 +/- 188 ml/m, r = 0.76) as well as Filtration Fraction (17.7 +/- 8.3 vs. 16.6 +/- 6.6%) were well correlated. These data show that within the normal range of GRF and RPF the method determining only plasma levels is as reliable as the standard method, giving the advantage to be less troublesome for patients and staff, requiring no bladder catheterization.

Extracellular volume, renal clearance and whole body permeability-surface area product in man, measured after single injection of polyfructosan.

In 22 patients non-compartmental analysis was applied to plasma disappearance curves obtained after a single injection of polyfructosan to measure volume of distribution (Vd), renal clearance (Cl) and the whole body permeability-surface area product (PdS) of the indicator. It was found that introduction of the theoretical plasma concentration at time zero, calculated as the injected amount divided by the plasma water volume independently determined, to the curves was necessary to determine the initial slope of the curve and subsequently calculate the PdS. This 'initial phase' approximation had negligible effect on the calculations of Vd and Cl. The approximation, however, allowed extension of the non-compartmental analysis to include calculation of the whole body PdS. The whole body PdS for polyfructosan was found to be 0.68 ml/100g X min or 429 ml/min. Assuming a capillary surface area of the whole human body of 50 cm2g-1, the average whole body diffusional permeability coefficient, Pd, for polyfructosan was found to be: 0.23 X 10(-5) cm X sec-1.

Comparison of plasma clearances of polyfructosan and 51Cr-EDTA in children.

Plasma clearances of polyfructosan and 51Cr-EDTA were determined in 38 children with renal diseases and in five healthy children in order to compare the values for the glomerular filtration rate (GFR) obtained by single injection procedures. Both one-compartment (Cr-EDTA-1) and two-compartment (Cr-EDTA-2) analyses were made of the isotope decay curve. The children were divided into two age groups: (a) 24 children below 6 years, and (b) 19 children between 8 and 16 years of age. In both groups Cr-EDTA-1 GFR and Cr-EDTA-2 GFR correlated significantly with polyfructosan GFR. The correlation coefficients, however, were somewhat lower in the younger (r = 0.74, r = 0.73) than in the older children (r = 0.91, r = 0.93). There were no significant differences between simultaneously obtained polyfructosan GFR and Cr-EDTA-2 GFR based on paired observations while Cr-EDTA-1 GFR was somewhat higher (16%) than the simultaneously obtained polyfructosan GFR. We conclude that estimates of GFR from polyfructosan and Cr-EDTA clearances based on two compartment analyses and single injection procedures are interchangeable.

Blood levels of high-molecular levan in mice as a function of the route and duration of administration.

The influence of duration and of various routes of administration of native levan on blood levels of the drug was studied in mice. The blood levels varied with the doses administered. With a single i.p. injection of 20 mg levan/mouse the blood levels rose more rapidly than with higher and lower doses. In the second and third week of daily injections the rise and fall in blood levels became less pronounced. With treatment longer than 3 weeks spikes of maximal concentrations reappeared. After s.c. injections blood levels rose more slowly than after i.p. treatment. The findings indicate that: 1. high doses slow down absorption of levan; 2. after 7-21 days of treatment the RES (reticulo endothelial system) is more active in removal of levan from the blood stream than at the beginning of the treatment; 3. after treatment of 3 weeks and more the RES appears to be blocked and is again slow in the uptake of levan from the blood stream.

[Determination of glomerular and tubular clearance in children without urine collection]. / Détermination de la clearance glomérulaire et tubulaire de l'enfant sans recueil d'urines

The determination of glomerular and tubular clearance in a child was carried out by using a method excluding urine collection, with a continuous intravenous infusion of polyfructosan and para-amino-hippuric acid. A loading injection followed by a continuous perfusion provides a constant plasmatic level 150 minutes after the beginning of the study.