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.

A sensitive and specific HPGPC-FD method for the study of pharmacokinetics and tissue distribution of Radix Ophiopogonis polysaccharide in rats.

Interest in antimyocardial ischemic activity of a graminan-type fructan with a weight average molecular weight of 4.8 kDa extracted from Radix Ophiopogonis (ROP) has necessitated the study of its pharmacokinetics and tissue distribution. For that, a simple HPGPC-FD method was developed for the sensitive and specific determination of FITC-ROP (fluorescein-isothiocyanate-labeled ROP) in plasma and rat tissues (heart, liver, spleen, lung, kidney, brain and stomach). The analyte was separated on a Shodex Sugar KS-802 high-performance gel column with 0.1 M phosphate buffer (pH 7.0) as mobile phase at a flow rate of 0.5 mL/min, and fluorescence detection at lambda(ex) 495 nm and lambda(em) 515 nm. The calibration curve for FITC-ROP was linear over the range 0.25-20.0 or 50.0 microg/mL in all studied biosamples with correlation coefficients > 0.995. The inter-day and intra-day precisions of analysis were not more than 10%, and assay accuracy ranged from 93 to 105% for plasma and from 89 to 108% for tissue homogenates. This method has been confirmed here to be suitable for the study of pharmacokinetics and tissue distribution of ROP and the achieved results are highly instructive for the further pharmaceutical development of ROP, suggesting the promising application of the method to the increasingly important carbohydrate-based drugs.

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 clearance of polyfructosan and extracellular body fluid distribution in idiopathic dilated cardiomyopathy and after heart transplantation.

The total extracellular fluid volume and distribution in plasma and interstitial spaces, and the microvascular permeability properties were studied in 16 nonedematous patients with congestive heart failure (CHF) due to idiopathic dilated cardiomyopathy and 17 such patients who underwent heart transplantation (HT) by analyzing the 3-hour plasma disappearance curve of polyfructosan. Eighteen healthy subjects served as controls. Polyfructosan (3.5 kD) is an extracellular marker and inulin analog transported almost solely by diffusion. The initial capillary membrane plasma clearance (i.e., the permeability-surface area product), the interstitial plasma clearance determined at 10 minutes (clearance[10), and the extracellular volume were determined from the polyfructosan curves. I-131-albumin was used as a plasma volume reference. Permeability-surface area product was elevated in both patient groups (6.6 +/- 1.9 ml/ kg/min in the CHF group and 6.7 +/- 2.0 ml/kg/min in the HT group vs 5.1 +/- 1.3 ml/kg/min in controls, p <0.01 for both), whereas clearance(10) was normalized in the HT group (4.5 +/- 0.9 ml/kg/min in the HT group, 4.4 +/- 0.7 ml/kg/min in controls vs 5.0 +/- 0.9 ml/kg/ min in the CHF group, p <0.1 and p <0.05, respectively). The normalization of interstitial plasma clearance of polyfructosan was associated with time since HT (r = 0.49, p <0.05). Plasma volumes were similar in all 3 groups (41 +/- 8 ml/kg in controls, 44 +/- 13 in the CHF group and 39 +/- 8 in the HT group). In contrast, total extracellular volume was elevated in both patients groups (177 +/- 27 ml/kg in the CHF group and 173 +/-27 in the HT group vs 152 +/- 12 in controls, p <0.01). The results strongly suggest a microvascular permeability defect in both patient groups that perhaps plays a role in the extravascular distribution of the excess extracellular fluid volume.

Effect of glandular kallikrein on distal nephron HCO3- secretion in rats and on HCO3- secretion in MDCK cells.

Renal kallikrein is localized in the connecting tubule cells and secreted into the tubular fluid at late distal nephron segments. The present experiments were performed to further test the hypothesis that renal kallikrein reduces bicarbonate secretion of cortical collecting duct (CCD). The effect of orthograde injections of pig pancreatic kallikrein (1 or 3 micrograms/ml) into the renal tubular system was investigated. Urine fractions (Fr) were collected after a 2-min stop flow. Changes in the urine fraction with respect to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/ Fr:Ff polyfructosan) increased significantly in the first two urine fractions collected after glandular kallikrein administration (kallikrein, 1 microgram/ml, P < 0.05; kallikrein, 3 micrograms/ml, P < 0.01). HCO3- secretion of collecting ducts was significantly reduced dose dependently by orthograde and also reduced by retrograde pig pancreatic kallikrein administration. Release of kinins into the fractions was not affected by the retrograde kallikrein injection, even though the kallikrein activity increased considerably (2.26 +/- 0.2 vs. 1.55 +/- 0.2, P < 0.05). Adequacy of retrograde injections for delivering substances to the CCD was demonstrated by injecting colloidal mercury and detecting the appearance of this mercury in the renal cortex by transmission electron microscopy. The integrity of the renal tissue after a retrograde ureteral injection was confirmed by scanning electron microscopy. These results confirm and extend previous data (M. Marin-Grez and P. Vallés. Renal Physiol. Biochem. 17: 301-306, 1994; and M. Marin-Grez, P. Vallés, and P. Odigie. J. Physiol. 488: 163-170, 1995) showing that renal kallikrein reduces bicarbonate secretion at the CCD, probably by inhibiting HCO3- transported by a mechanism unrelated to its kininogenase activity. Support for this assessment was obtained in experiments testing the effect of kallikrein on the luminal bicarbonate secretion of a subpopulation of Madin-Darby canine kidney cells capable of extruding the anion. Kallikrein inhibited HCO3-/Cl- exchange, and the degree of inhibition was dose dependent. This inhibition occurred in the absence of kininogen in the bathing solution.

Predicting glomerular function from adjusted serum creatinine in renal transplant patients.

In 22 individuals with a renal graft the correlations between the renal clearance of polyfructosan (CLPF), renal creatinine clearance (CLcr)--established under the same conditions as CLPF--and the value of glomerular function predicted using the equation by Cocroft and Gault (PredCLcr) were followed up, at an interval of 2-3 months, for 8-22 months. A significant linear correlation (r = 0.777, p < 0.001) was found between PredCLcr and CLPF as well as between PredCLcr and CLcr (r = 0.801, p < 0.001). Equally significant correlations, however, were established when relating the serum concentrations of creatinine (Scr) to 1/CLPF (r = 0.784, p < 0.001) and Scr to 1/CLcr (r = 0.744, p < 0.001). The values of the PredCLcr/CLPF and PredCLcr/CLcr ratios during follow-up in one and the same individual may vary significantly. This fluctuation exceeds maximal error of the analytical methods employed in one third of the individuals examined. When considering stabilization or slow changes in graft function on the basis of PredCLcr and CLPF we found significant discrepancies in more than one half of the individuals examined (64%). The findings support the assumption that more accurate methods must be used to assess graft glomerular function on long-term follow-up.

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.

A reappraisal of the measurement of glomerular filtration rate in pre-term infants.

Thirty-seven single-injection polyfructosan-S (PF-S, Inutest) and 98 continuous-infusion PF-S/creatinine clearance studies were performed in 39 sick very low birth weight infants. The single-injection clearance method for measuring glomerular filtration rate has been shown to be a reliable technique if sampling is continued for 8 h or more and the PF-S (Inutest) assay is sensitive, accurate and precise. The continuous-infusion clearance method is also valid if the infusion is continued for more than 24 h and preceded by a loading dose in the form of a double-rate infusion for 8 h. Creatinine clearance is usually less than PF-S clearance, the mean ratio being 0.91, suggesting that there is some creatinine reabsorption in the renal tubule in sick very low birth weight infants.

The glomerular filterability of polyfructosan-S in immature infants.

Gel chromatography was performed on 21 plasma, 5 urine and 3 spinal fluid samples from pre-term infants who had received intravenous polyfructosan-S (PF-S), in order to investigate whether all sizes of PF-S molecule are filtered at the glomerulus and penetrate deep extra-cellular fluid compartments. Plasma obtained after 24 h continuous infusion of PF-S had identical chromatograms to the aqueous solution, suggesting no glomerular hold-up of the largest molecules. PF-S had a broader chromatogram than inulin, suggesting that it has larger molecules. Urine and plasma samples obtained early after a single injection had an excess of high molecular weight polyfructoside, suggesting that small molecules penetrate deep extracellular fluid compartments more easily. Spinal fluid samples had an excess of low molecular weight polyfructoside, suggesting slow penetration of the largest molecules.

[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.