Milk ribonuclease-enriched lactoferrin induces positive effects on bone turnover markers in postmenopausal women.

UNLABELLED: Current treatments for postmenopausal osteoporosis suffer from side effects. Safe and natural milk proteins, ribonuclease, and lactoferrin promote formation of new capillaries and bone formation. A ribonuclease-enriched lactoferrin supplement studied here, demonstrates significant reduction in resorption and increase in formation, towards restoring the balance of bone turnover within 6 months. INTRODUCTION: Osteoporosis, a major health issue among postmenopausal women, causes increased bone resorption and reduced bone formation. A reduction in angiogenesis could also contribute to this imbalance. Current treatments such as hormone replacement therapy and bisphosphonates have drawbacks of severe side effects. Milk ribonuclease (RNase) is known to promote angiogenesis and lactoferrin (LF) to stimulate bone formation by osteoblasts. We examine the effect of ribonuclease-enriched lactoferrin supplement on the bone health of postmenopausal women. METHODS: A total of 38 healthy, postmenopausal women, aged 45 to 60 years were randomized into placebo or RNAse-enriched-LF (R-ELF) supplement groups. The bone health status was monitored by assessing bone resorption markers, serum N-telopeptides (NTx), and urine deoxypyridinoline (Dpd) crosslinks and serum bone formation markers, bone-specific alkaline phosphatase (BAP), and osteocalcin (OC). RESULTS: R-ELF supplementation demonstrated a decrease in urine Dpd levels by 14% (19% increase for placebo) and serum NTx maintained at 24% of the baseline (41% for placebo), while serum BAP and OC levels showed a 45% and 16% elevation (25% and 5% for placebo). CONCLUSIONS: R-ELF supplementation demonstrated a statistically significant reduction in bone resorption and increase in osteoblastic bone formation, to restore the balance of bone turnover within a short period.

Inhibition of HIV-1 in monocyte/macrophage cultures by 2',3'-dideoxycytidine-5'-triphosphate, free and in liposomes.

The antiviral effects of 2',3'-dideoxycytidine (ddC), 2',3'-dideoxycytidine-5'-triphosphate (ddCTP) and liposome-encapsulated ddCTP [L(ddCTP)] were compared in cultured human monocyte-macrophages (M/M) infected with HIV-1. These treatments inhibited virus replication at nanomolar drug levels with activities in the order ddC greater than ddCTP = L(ddCTP). Studies on drug stability and uptake suggest that a large part of the free ddCTP is dephosphorylated before entering the cells, whereas L(ddCTP) remains stable over days and is taken up, probably by endocytosis. The response to L(ddCTP) suggests that the capability of liposomes for targeting drugs to macrophages in vivo could potentially be exploited to improve the therapeutic index of dideoxynucleoside drugs.

Effect of dipyridamole on transport and phosphorylation of thymidine and 3'-azido-3'-deoxythymidine in human monocyte/macrophages.

Dipyridamole (DPM), a commonly used coronary vasodilator and antithrombotic drug, was shown recently to potentiate the antiviral effect of 3'-azido-3'-deoxythymidine (AZT) in HIV-1 infected human monocyte-derived macrophages (M/M) in vitro. We report in the present study that in uninfected M/M, DPM markedly inhibited cellular uptake of [3H]thymidine (dThd) and its incorporation into the nucleotide pools, particularly the dThd-triphosphate pool. In contrast, DPM did not affect cellular uptake and phosphorylation of [3H]AZT. Since dThd counteracts the phosphorylation and antiviral action of AZT, these findings support the hypothesis that the potentiation of the anti-HIV effect of AZT is due, at least in part, to differential inhibition of nucleoside salvage.

Transdermal iontophoretic delivery of enkephalin formulated in liposomes.

Transdermal iontophoretic transport of a liposomal formulation of [Leu5]enkephalin, across human cadaver skin, was investigated. Franz (vertical) cells were supplied with 0.5 mA/cm2 current density via silver/silver chloride electrodes from a Scepter power supply. Enkephalin spiked with [3H]enkephalin was transported across skin from anode or cathode, depending on the charge on the molecule. Liposomes or their constituents were shown to penetrate into the skin. Enkephalin, when delivered iontophoretically at its isoelectric point, from liposomes carrying positive or negative charge on their surface, resulted in permeation of radioactivity which was same or less than that of the controls when analyzed by liquid scintillation counting. When analyzed by radiochromatography detector on HPLC, degradation of enkephalin during transport was observed, with several degradation peaks in the chromatogram. The degradation was less in liposome formulations, as compared to controls. This is the first report of the combined use of liposomes and iontophoresis for transdermal delivery.

Partitioning and thermodynamics of dipyridamole in the n-octanol/buffer and liposome systems.

The thermodynamics of partitioning (K) of dipyridamole has been determined in n-octanol/buffer and liposome-buffer systems at pH 7.4. Dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) were used to prepare multilamellar liposomes. Partitioning of dipyridamole did not depend on the amount of n-octanol employed, however, partitioning was dependent upon the quantity of DMPC employed to prepare liposomes. Plots of log K vs 1/T were linear in the n-octanol and liposome systems. Partitioning was generally greater in liposomes than in the n-octanol/buffer system. Among liposomes, the partitioning was greater in DMPC liposomes at all temperatures. The values of enthalpy (delta H) and entropy (delta S) were positive in both the n-octanol and liposome systems. These values were lower in DMPC liposomes and were comparable in the n-octanol and DPPC liposomes. Thus, the interaction of dipyridamole depends on the rigidity of lipid bilayers and liposomes constitute a more selective partitioning system than the n-octanol/buffer system.

Encapsulation, stability and in-vitro release characteristics of liposomal formulations of colchicine.

The severe toxicity and low therapeutic index of colchicine limit its therapeutic use. Encapsulation in liposomes might reduce these toxic effects. The objective of this study was to determine the factors influencing encapsulation of colchicine in liposomes and to optimize the encapsulation parameters. Colchicine was encapsulated in multilamellar liposomes and large unilamellar liposomes prepared using various phospholipids. The effects of method of preparation, type of vesicle, charge, and concentration of cholesterol on encapsulation of colchicine in liposomes were investigated. Also, stability of colchicine under stress conditions and at various temperatures, and in-vitro release characteristics were determined. A significant difference in encapsulation of colchicine in multilamellar liposomes was observed when prepared by two different methods. Induction of charge on the liposome surface increased encapsulation of colchicine in multilamellar liposomes, but did not affect large unilamellar liposomes. The liposome preparations could withstand simulated transport conditions and frequent changes in temperature. Particle size and concentration of colchicine did not change significantly during storage at various temperatures for six months. In order to retain encapsulated colchicine in liposomes, storage at or below room temperature was found to be suitable. In-vitro release of colchicine from large unilamellar liposomes was biphasic and was influenced by two rate-limiting barriers, the dialysis membrane and the liposome bi-layers. For optimum encapsulation and stability of colchicine liposomes were prepared from a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and either stearylamine or dicetyl phosphate.

Comparative study of separation of non-encapsulated drug from unilamellar liposomes by various methods.

The purpose of this study was to compare the various methods available to separate non-encapsulated drug from large unilamellar liposomes (LUV). Multilamellar liposomes (MLV) were prepared by thin film hydration using distearoylphosphatidylcholine:cholesterol (2:1 molar ratio). MLVs were passed through a 0.2 micron polycarbonate membrane using an extruder to prepare LUVs. Particle size of liposome preparations was characterized using a submicron particle-size analyser. The non-encapsulated drug was separated by: filtering through Centrifree tubes; passing through gel (Sepharose-4B and Sephadex G-25M); passing through minicolumn; ficoll density gradient; protamine aggregation; or dialysis. The dialysis method was found to be unsuitable for separation of non-encapsulated drug due to equilibration of encapsulated drug as the free drug was dialyzed. The upper limit for lipid concentration was 5 mg mL-1 using the Centrifree method. Separation using gel chromatography led to dilution of liposome preparation. Minicolumn and density gradient techniques did not lead to sample dilution, however the minicolumn method was tedious. The time required for separation of liposomes by protamine aggregation was longer for neutral liposomes. Thus it was concluded that the Centrifree was the fastest method to estimate encapsulation; the density gradient method was ideal to separate non-encapsulated drug; and protamine aggregation was the least expensive method to estimate encapsulation efficiency.

Fc-receptor-mediated targeting of antibody-bearing liposomes containing dideoxycytidine triphosphate to human monocyte/macrophages.

Liposomes bearing surface-attached antibody (L-Ab) molecules can be used for various purposes including the immunospecific delivery of drugs or other materials to antigenic target cells. In this study, L-Ab were prepared to deliver an anti-human immunodeficiency virus (HIV) drug, dideoxycytidine triphosphate (ddCTP) to human monocyte/macrophages. Cells of the monocyte/macrophage lineage are an important reservoir of HIV-1. A mouse monoclonal antibody IgG2a was labelled with 125I and modified using N succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as a heterobifunctional reagent in order to conjugate with liposomes to produce a covalent bond (thioether). SPDP-modified antibody was incubated with liposomes containing 5 mol% of maleimido phenyl butyrate phosphatidylethanolamine (MPB-PE) at room temperature (21 degrees C) for 24 h. L-Ab were separated from free and aggregated antibodies by centrifugation. L-Ab were characterized by measuring particle size and binding to anti-mouse IgG-sepharose. Ninety five per cent of the liposomal (L-Ab) lipid label was bound to anti-mouse IgG-sepharose, whereas only 7% of plain liposomes were bound, indicating non-specific binding. Uptake of L-Ab was measured in human monocyte/macrophages as a function of time and compared with that of plain liposomes. The uptake increased with time and it was 4-6 times greater than that of plain liposomes although part of that effect may have been due to unreacted MPB groups.

Formulation, characterization, and in vitro release of glyburide from proliposomal beads.

The objective of our study was to formulate and evaluate proliposomes in the form of enteric-coated beads using glyburide as a model drug. The beads were enteric coated with Eudragit L-100 by a fluidized bed coating process using triethyl citrate as plasticizer. Content uniformity of glyburide was estimated using HPLC analysis of beads dissolved in methanol. These proliposomal beads formed liposomes on disintegration in phosphate buffered saline (pH 7.4), which was confirmed by transmission electron microscopy. The dissolution study of enteric-coated beads exhibited enhanced dissolution compared with pure drug and a marketed product. Liposomes can be successfully prepared for oral administration in the form of enteric-coated beads that may offer a stable system to produce liposomes for oral administration.

Liposomal formulation of mirfentanil hydrochloride.

Mirfentanil hydrochloride, a novel CNS analgesic with a short duration of action, was successfully encapsulated in liposomes having a variety of compositions. The lipid composition of the formulation was varied to optimize the stabilization of liposomes and the encapsulation of solutes. Retention of mirfentanil hydrochloride was evaluated by storing loaded liposomes at several temperatures, and also after the physical stressing of formulations. High efficiency of drug encapsulation was observed in liposomes prepared using dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) and the ternary mixture of dimyristoyl-L-alpha-phosphatidylcholine, cholesterol, and dicetyl phosphate (DMPC/CHOL/DCP), both with and without the further incorporation of monosialoganglioside (GM1). Only 35% of encapsulated drug was lost when the formulations containing GM1 were incubated with human plasma over a 24 hour period, suggesting that liposomal formulations containing GM1 could be used to control drug release in vivo.

Correlation of partitioning of nitroimidazoles in the n-octanol/saline and liposome systems with pharmacokinetic parameters and quantitative structure-activity relationships (QSAR).

The partitioning of a series of nine nitroimidazole drugs in liposomes (log Km) of various compositions has been determined and compared to their partitioning in the n-octanol/saline system (log K) at 30 degrees C. The log Km ranged from 1.5 to 0.5 and was three- to fourfold greater than the log K; further, the linear correlation coefficient was greatest when cholesterol (CHOL)-free liposomes were used. Functional-group contributions were compared from their hydrophobic substituent constants and, except in the case of RO-07-2044 and iodoazomycin riboside, yielded negative values in all systems. Literature values of four pharmacokinetic parameters obtained in dogs and acute LD50 values of the nitroimidazoles in BALB/c mice were highly correlated with log K or log Km only in CHOL-free liposomes. Comparing the relative sensitizing effect of the nitroimidazoles in murine EMT-6 or Chinese hamster V79 tumor cell cultures and their partition coefficients, the correlation in EMT-6 cells was poor, whereas the correlation in V79 cells was greater than 0.9 when log Km was used but less than 0.6 when log K was used. Thus, the liposome model is a better predictor of nitroimidazole activity than the n-octanol/saline system and, also, it is a more flexible model for selecting the optimum conditions for QSAR studies.

Factors affecting microencapsulation of drugs in liposomes.

Liposomes have been used as carriers for drugs, toxins, enzymes, proteins/peptides and other bioactive materials there are several liposomal formulations that are being investigated in preclinical and clinical trials. Achieving high encapsulation as well as retention of the encapsulated drug is very important in developing liposomes as drug carriers. A high drug-to-lipid ratio is likely to reduce the cost of formulations and also the risk of lipid-induced toxicity following their injection. Comparison of the encapsulation efficiency of the drug in liposomes with the therapeutic dose indicates whether, in principle, liposomes can be used as a delivery system for that drug. The optimization of the liposomal encapsulation of a drug is usually based on trial and error, rather than on a thorough investigation of the factors affecting it. To obtain optimum encapsulation of a drug into a liposomal preparation, parameters influencing both the liposome and the drug need to be carefully considered during the early stages of development. In this review, factors that affect encapsulation of drugs in liposomes such as liposome size and type, charge on the liposome surface, bilayer rigidity, method of preparation, remote loading, addition of ion pairing, and complexing agents and characteristics of the drug to be encapsulated are discussed.

Oral sustained-release bioadhesive tablet formulation of didanosine.

The objective of this study was to formulate a hydrogel-forming bioadhesive drug delivery system for oral administration of didanosine (ddI). The aim of this tablet dosage form is to improve the oral absorption of ddI by delivering it in small doses over an extended period and localizing it in the intestine by bioadhesion. Compressed tablets of ddI using Polyox WSRN-303, Carbopol 974P-NF, and Methocel K4M as the bioadhesive release rate-controlling polymers were prepared. The effect of polymer concentration on the release profile and in vitro bioadhesion of the matrix tablets was studied. Tablet formulations with Polyox WSRN-303 (10%) and Methocel K4M (30%) showed 93 and 90% drug release, respectively, after 12 h. The drug release was found to be linear when fitted in the Higuchi equation (square-root time equation), suggesting zero-order release. Carbopol 974-P-NF was found to inhibit the complete release of ddI because of drug-polymer interaction; hence, is not suitable for formulation of ddI. Drug diffusion and swelling of the polymer (anomalous Fickian release) was found dominant in ddI release. In general, in vitro bioadhesion increased with an increase in polymer concentration. Tablets containing a single polymer can be designed to form hydrogels serving the dual purpose of bioadhesion and sustained release.

Solubilization of liposomes by weak electrolyte drugs. I. Propranolol.

The solubilization of dimyristoylphosphatidylcholine (DMPC) liposomes by a weak electrolyte drug, propranolol (PPL) hydrochloride, has been studied as a function of pH, [PPL], [DMPC], and temperature. The solubilization of liposomes at 40 degrees C by 0.2 mM PPL occurred at different rates from 2.9 to 14.4 mM DMPC but converged at complete solubilization after 13 hr at pH 12.0. At the same [PPL], solubilization was complete after 18 days at pH 11.0, but incomplete solubilization occurred at pH 10.0 and not at all at lower pH's. In 14.4 mM DMPC liposomes, solubilization was gradual and proportional to the [PPL] from 0.001 to 0.10 mM up to 95 hr, then rapid thereafter. The [PPL] at which the solubilization efficiency began to increase rapidly was determined to be 0.078 mM. The rate of solubilization was also influenced by the fluidity of the bilayers, a sevenfold increase in the time for complete solubilization being observed upon cooling from 40 to 20 degrees C. Surface tension (st) data confirmed a low critical micelle concentration (CMC) and continued decrease in the st above the CMC. It is concluded that the critical ratio of PPL to DMPC for solubilization occurs in localized regions of the bilayers, with total solubilization at different rates depending on the [PPL] and the physical properties of the liposomes. The processes may be used advantageously to prepare small vesicles or to extract lipids or proteins, more efficiently than detergents, from biological membranes.

NMR study of the interaction of beta-blockers with sonicated dimyristoylphosphatidylcholine liposomes in the presence of praseodymium cation.

The interaction of a series of beta-adrenoreceptor blocking agents with unilamellar dimyristoylphosphatidylcholine (DMPC) liposomes has been studied by proton nuclear magnetic resonance (1H-NMR) in the presence of praseodymium cation (Pr3+) at 30 degrees C. Addition of Pr3+ increased the splitting of the trimethylammonium group signals arising from the phospholipid molecules located at the internal and external surfaces of the bilayers. Adding Pr3+ caused a considerable downfield shift of the external peak but only a slight upfield shift of the internal peak (approximately 3%). The difference in chemical shift of the external and internal peaks (delta Hz) increased linearly as a function of Pr3+ concentration up to 10 mM. The addition of beta-blockers reversed the effect of Pr3+, and propranolol exerted the most pronounced effect, causing complete reversal of the splitting at a concentration of 5 mM. Much higher concentrations of other beta-blockers were required to displace Pr3+. A linear correlation between Pr3+ displacement (P) and logarithm of the apparent partition coefficient (K'm) in DMPC liposomes was obtained for hydrophobic beta-blockers, but hydrophilic beta-blockers did not fit this correlation. It appears that beta-blockers that have ortho or meta substitution require penetration of the liposome bilayers before significant polar group interaction can occur. On the other hand, beta-blockers that have para substitution and low K'm values are able to interact with the polar surfaces of the liposomes without penetration to cause displacement of Pr3+.

Enhancement of dissolution of ethopropazine using solid dispersions prepared with phospholipid and/or polyethylene glycol.

The purpose of this study was to improve the dissolution properties of a poorly water soluble and bioavailable drug, ethopropazine HCl (ET), by incorporating the drug in three different types of solid dispersion systems. Solid dispersions of ET were prepared using 1:1 (w/w) ratios of (1) phospholipid (1,2 dimyristoyl-sn-glycerophosphocholine) (DMPC), (2) polyethylene glycol 8000 (PEG8000), and (3) a novel combination of both DMPC and PEG8000. Using the solvent method of preparation, ET and DMPC and/or PEG were dissolved in chloroform, and solvent subsequently was evaporated using nitrogen gas. The resulting solid dispersion(s) was passed through a 60-mesh sieve. Characterization of ET/DMPC solid dispersion was performed by differential scanning calorimetry (DSC) and X-ray diffractometry studies. Dissolution studies conducted in phosphate buffered saline (PBS) (pH 7.4, 37 degrees C +/- 0.5 degrees C) using the USP type II (paddle) dissolution apparatus showed significant increases in the dissolution rate of ET with all the solid dispersions in this study. Specifically, within the first 5 min (D5), solid dispersions containing ET/DMPC (1:1) showed an eightfold increase in dissolution; in combination with DMPC and PEG8000 (1:1), there was an approximately sixfold increase; and a fourfold increase was observed with PEG8000 (1:1). Complete dissolution of all solid dispersions occurred within 60 min (D60) of the run. Storage of the ET/DMPC sample for over 4.5 months revealed a decrease in the dissolution rate when compared to freshly prepared sample. Overall, it was concluded that the dissolution rate of ET significantly improved when dispersed in all the selected carrier systems. However, the solid dispersion of ET/DMPC was observed to be superior to the other combinations used.

Solubilization of multilamellar liposomes in the presence of non-ionized drug.

The solubilization of multilamellar liposomes by metoprolol tartrate (MPL) has been studied as a function of pH, [MPL], [dimyristoylphosphatidylcholine (DMPC)], temperature and lipid composition. The solubilization of liposomes at 37 degrees C by 7.3 nM MPL occurred at different rates at different pH values. MPL completely solubilized by 7.2 mM DMPC liposomes after about 17 h at pH 12, but only a partial solubilization occurred at pH 10 and 11. Between pH 7 and 9 no change in turbidity was observed after 1 week. Addition of cholesterol (CHOL) to DMPC (2:1 mol) had very little effect on solubilization after 24 h, however with DMPC:CHOL (5:1 mol) the decrease in turbidity was observed after 24 h, even though solubilization was much less compared with that of DMPC alone. The rate of solubilization was decreased when dipalmitoylphosphatidylcholine liposomes were employed. Addition of dicetylphosphate (DCP) to DMPC liposomes reduced the rate of solubilization significantly. The solubilization of liposomes by 7.3 mM MPL as a function of [DMPC], indicated that the lower the liposome concentration the greater the effect on solubilization. It is concluded that MPL in the non-ionized form has a solubilizing effect on liposomes, and addition of CHOL or DCP to DMPC has a stabilizing effect against solubilization.

The stability of amphotericin B in a mixture of fungizone and optimoist.

The physical and chemical stability of amphotericin B in a mixture of Fungizone and Optimoist was studied. The mixture was prepared by mixing equal volumes of Fungizone and Optimoist. The rate of sedimentation of amphotericin B was evaluated in Fungizone, Fungizone:Optimoist (1:1) and Fungizone:distilled water (1:1) mixture. The chemical stability of amphotericin B in a mixture of Fungizone:Optimoist (1:1) was determined by high-performance liquid chromatography from samples stored at different temperatures. The rate of sedimentation was faster when Fungizone was mixed with distilled water as compared to Optimoist. Amphotericin B was stable for three weeks stored in the refrigerator (4 deg C) and for three weeks at room temperature (25 deg C). Fungizone can be diluted with Optimoist for topical application. The mixture should be shaken well before administration in order to disperse any sediment.