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Biomedical subjects

F M Wong

Publications and source records attributed to F M Wong.

15 recordsLinked to original sources
PubMed2000

Use of poly(ethylene glycol)-lipid conjugates to regulate the surface attributes and transfection activity of lipid-DNA particles.

We evaluated the use of poly(ethylene glycol) (PEG)-modified lipids to control the surface properties of a lipid-based gene transfer system. The lipid-DNA particles (LDPs) used form spontaneously when plasmid DNA is added to mixed detergent lipid micelles consisting of the non-ionic detergent n-octyl-D-glucopyranoside, the cationic lipid dioleyldimethylammonium chloride (DODAC), the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and selected PEG-modified phosphatidylethanolamines. The inclusion of DODAC is required to form the hydrophobic lipid-DNA complex. DOPE is included to facilitate dissociation of DNA from the cationic lipid and the PEG-modified lipids are added in an effort to stabilize the surface attributes of the resulting lipid-DNA particles. We used PEG-lipids that varied in acyl chain composition because of recent results demonstrating acyl chain dependent transfer of PEG-lipids from lipid vesicles, providing the potential to allow a transformation of the surface properties due to loss of surface grafted PEG. The addition of PEG-modified lipids does not interfere in LDP formation and its presence favors formation of smaller particles (75 nm in contrast to 130 nm in the absence of the PEG-modified lipid). PEG-lipid incorporation causes a concentration dependent reduction in LDP-mediated transfection of B16/BL6 melanoma cells, a result that can be partially attributed to a reduction in particle binding to cells. However, significant LDP binding to B16/BL6 cells was still observed under conditions where LDP transfection activity was reduced by more than 85%. The potential for PEG to interfere with LDP processing following cell binding is discussed.

Animals↗
PubMed2000

Eradication of human non-Hodgkin's lymphoma in SCID mice by BCL-2 antisense oligonucleotides combined with low-dose cyclophosphamide.

Cancers overexpressing Bcl-2 protein, which prevents programmed cell death (apoptosis), are less sensitive to stresses that produce cellular damage, including chemotherapy. If the level of Bcl-2 protein can be reduced sufficiently using antisense oligonucleotides (ASOs) targeting the gene message, then cytotoxic agents may be rendered more effective in eliminating disease and increasing cure rate. Preclinical studies in SCID mice bearing Bcl-2 overexpressing systemic human B-cell lymphoma (DoHH2) were undertaken to support development of a clinical trial. These data confirm that a combination of an ASO (5 mg/kg) targeting bcl-2 and a low dose of cyclophosphamide (35 mg/kg) was an effective strategy, leading to the eradication of the DoHH2 cells in vivo and cure of the animals. When mice deficient in natural killer cell activity were treated with an ASO, similar results were observed, suggesting that ASO stimulation of the host immune system was not a significant factor in elimination of lymphoma cells. These studies indicate that therapeutic strategies involving the use of an ASO targeting bcl-2 in combination with a cytotoxic agent may improve clinical outcomes.

Animals↗
PubMed1999

Electrostatically mediated interactions between cationic lipid-DNA particles and an anionic surface.

In an effort to model the interaction of lipid-based DNA delivery systems with anionic surfaces, such as a cell membrane, we have utilized microelectrophoresis to characterize how electrokinetic measurements can provide information on surface charge and binding characteristics. We have established that cationic lipids, specifically N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC), incorporated into liposomes prepared with 1, 2-dioleoyl-i-glycero-3-phosphoethanolamine (DOPE) or 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 50 mol%, change the inherent electrophoretic mobility of anionic latex polystyrene beads. Self-assembling lipid-DNA particles (LDPs), prepared at various cationic lipid to negative DNA phosphate charge ratios, effected no changes in bead mobility when the LDP charge ratio (+/-) was equal to or less than 1. Increasing the LDP concentration in a solution of 0.1% (w/v) anionic beads resulted in a charge reversal effect when a net charge of LDP to total bead charge ratio (+/-) of 1:1 was observed. LDP formulations, utilizing either DOPE or DOPC, showed similar titration profiles with a charge reversal observed at a 1:1 net LDP to bead charge ratio (+/-). It was confirmed through centrifugation studies that the DNA in the LDP was associated with the anionic latex beads through electrostatic interactions. LDP binding, rather than the binding of dissociated cationic lipids, resulted in the observed electrophoretic mobility changes of the anionic latex beads.

Anions↗
PubMed1998

Comparison of different hydrophobic anchors conjugated to poly(ethylene glycol): effects on the pharmacokinetics of liposomal vincristine.

Poly(ethylene glycol) (PEG) conjugated lipids have been used to increase the circulation longevity of liposomal carriers encapsulating therapeutic compounds. PEG is typically conjugated to distearoylphosphatidylethanolamine (DSPE) via a carbamate linkage that results in a net negative charge on the phosphate moiety at physiological pH. It was anticipated that the presence of this negative charge could have deleterious effects on liposome pharmacokinetic characteristics. We describe here the synthesis of a new class of neutrally charged PEG-lipid conjugates in which the PEG moiety was linked to ceramide (CER). These PEG-CER conjugates were compared with PEG-DSPE conjugates for their effects on the pharmacokinetics of liposomal vincristine. PEG-CER (78% palmitic acid, C16) and PEG-DSPE achieved comparable increases in the circulation lifetimes of sphingomyelin/cholesterol (SM/chol) liposomes. However, PEG-DSPE significantly increased the in vitro and in vivo leakage rates of vincristine from SM/chol-based liposomes compared to vincristine leakage observed when PEG-CER was used. The increase in drug leakage observed in vitro that was due to the presence of PEG-DSPE was likely due to the presence of a negative surface charge. Analysis of the electrophoretic mobilities of these formulations suggested that the negative surface charges were shielded by approx. 80% by the PEG layer extending from the membrane surface. In contrast, formulations containing PEG-CER had no surface charge and no electrophoretic mobility. A comparison of the effects of the ceramide acyl chain length (C8 through C24) on the pharmacokinetics of SM/chol/PEG-CER formulations of vincristine demonstrated that longer acyl chains on the PEG-CER were associated with longer circulation lifetimes of the liposomal carriers and, consequently, higher plasma vincristine concentrations. These data suggest that the short chain PEG-ceramides underwent rapid partitioning from the vesicles after i.v. administration, whereas the longer chain PEG-ceramides had stronger anchoring properties in the liposome bilayers and partitioned slowly from the administered vesicles. These data demonstrate the utility of ceramide-based steric stabilizing lipids as well as the potential for developing controlled release formulations by manipulating the retention of the PEG-ceramide conjugate in liposome bilayers.

Animals↗
PubMed1998

Characterization of lipid DNA interactions. I. Destabilization of bound lipids and DNA dissociation.

We have recently described a method for preparing lipid-based DNA particles (LDPs) that form spontaneously when detergent-solubilized cationic lipids are mixed with DNA. LDPs have the potential to be developed as carriers for use in gene therapy. More importantly, the lipid-DNA interactions that give rise to particle formation can be studied to gain a better understanding of factors that govern lipid binding and lipid dissociation. In this study the stability of lipid-DNA interactions was evaluated by measurement of DNA protection (binding of the DNA intercalating dye TO-PRO-1 and sensitivity to DNase I) and membrane destabilization (lipid mixing reactions measured by fluorescence resonance energy transfer techniques) after the addition of anionic liposomes. Lipid-based DNA transfer systems were prepared with pInexCAT v.2.0, a 4.49-kb plasmid expression vector that contains the marker gene for chloramphenicol acetyltransferase (CAT). LDPs were prepared using N-N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and either 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). For comparison, liposome/DNA aggregates (LDAs) were also prepared by using preformed DODAC/DOPE (1:1 mole ratio) and DODAC/DOPC (1:1 mole ratio) liposomes. The addition of anionic liposomes to the lipid-based DNA formulations initiated rapid membrane destabilization as measured by the resonance energy transfer lipid-mixing assay. It is suggested that lipid mixing is a reflection of processes (contact, dehydration, packing defects) that lead to formulation disassembly and DNA release. This destabilization reaction was associated with an increase in DNA sensitivity to DNase I, and anionic membrane-mediated destabilization was not dependent on the incorporation of DOPE. These results are interpreted in terms of factors that regulate the disassembly of lipid-based DNA formulations.

Animals↗
PubMed1996

Cationic lipid binding to DNA: characterization of complex formation.

We recently demonstrated that cationic lipids, added in monomer or micellar form, bind to DNA, resulting in the formation of a hydrophobic complex. This complex can serve as a well-defined intermediate in the preparation of DNA-lipid particles (DLPs) with many potential applications for delivery of polynucleotides in vitro and in vivo. To develop a better understanding of the factors governing complex formation, we have characterized the cationic lipid/DNA binding reaction. This was evaluated by measuring DNA and cationic lipid (DODAC) complex formation using the Bligh and Dyer extraction procedure. Efficient recovery of DNA (> 95%) in the organic phase was achieved when sufficient monocationic lipids interact with DNA phosphate groups. The rate of binding depends on the amount of DNA or cationic lipid present in the system. The time required to generate the hydrophobic complex was increased when < 10 micrograms of DNA or < 40 nmol of DODAC was present. Surprisingly, the rate of complex formation was contingent on the incubation period after partitioning the DNA/lipid mixture into organic and aqueous phases. These results suggest that the cationic lipid/DNA complex forms at the aqueous/organic interface and that DNA/lipid binding is dependent on multivalent interactions at this interface. A Scatchard analysis of DNA/DODAC binding demonstrated that the binding reaction exhibits a high degree of positive cooperativity. The apparent dissociation constant (Kn), using data obtained under conditions where DODAC binding to DNA approached saturation, indicated a high-affinity reaction (Kn > 10(-11) mol L-1). At this point, approximately 8400 mol of DODAC was bound per mole of DNA, which is equivalent to a charge ratio (+/-) of 0.585 for the 7.2 kb plasmid used and suggests that formation of the hydrophobic complex occurs at a stage prior to charge neutralization. The influence of other lipids on DNA/cationic lipid binding at the aqueous/organic interface was also studied. Cholesterol and DOPC had little effect on DNA/DODAC binding while the anionic lipids LPI, DOPS, and DMPG inhibited complex formation. The zwitterionic lipid DOPE, however, had a concentration-dependent effect on cationic lipid binding that was also dependent on the mixing order. We believe that this approach for evaluating lipid/DNA binding provides an effective procedure for assessing factors which control the dissociation of lipids from DNA and may be beneficial in the selection of lipids for effective use in gene transfection studies.

Binding Sites↗
PubMed1987

Cimetidine transport in isolated luminal membrane vesicles from rabbit kidney.

Experiments were conducted to study the transport of the histamine H2-receptor antagonist, cimetidine, in luminal membrane vesicles prepared from rabbit renal cortex. Cimetidine accumulated in the vesicles with time. Cimetidine uptake was sensitive to changes in vesicle size, suggesting that the compound is transported into an osmotically reactive intravesicular space. Its rate of uptake could be described by both a saturable and a nonsaturable process. The Km was 4.6 +/- 4.0 microM and the Vmax was 6.8 +/- 2.3 pmol X s-1 X mg protein-1 (mean +/- SD, n = 4). N1-methylnicotinamide (NMN), cimetidine, cimetidine sulfoxide, and ranitidine inhibited the uptake of cimetidine. Cimetidine uptake in the presence of an outwardly directed proton gradient was enhanced in vesicles preloaded with a higher concentration of unlabeled cimetidine (2.4 X 10(-4) M). An outwardly directed proton gradient enhanced the uptake of cimetidine to values exceeding its equilibrium accumulation. Uptake stimulated in this way could be inhibited by the cation, NMN, the bases, ranitidine, and cimetidine sulfoxide, and interestingly, by the anion, probenecid. The effect of probenecid did not appear to be due to nonspecific effects on membrane binding, membrane potential, or vesicle size. These data are consistent with data obtained in isolated perfused proximal tubules, demonstrating that probenecid inhibits cimetidine transport. The data in this study suggest that the effect of probenecid on cimetidine transport specifically involves the transporter in the luminal membrane.

Animals↗
PubMed1987

Comparison of binding affinities and negative inotropic potencies of the 1,4-dihydropyridine calcium channel blockers in rabbit myocardium.

The binding and pharmacologic response of a series of 1,4-dihydropyridine analogs were examined in rabbit myocardium. [3H]Nitrendipine was used to label specific binding sites in myocardial membrane particulates and displacement experiments were carried out with the unlabeled analogs to determine their IC50 values. Binding of [3H]nitrendipine could be characterized by a Kd of 0.15 +/- 0.06 nM and a maximum number of binding sites of 247 +/- 150 fmol/mg of protein. Saturation binding experiments performed with higher concentrations of [3H]nitrendipine did not reveal the presence of a lower affinity site. Binding IC50 values of 12 unlabeled 1,4-dihydropyridine analogs ranged from 4.3 X 10(-10) M to 1.32 X 10(-6) M. The negative inotropic effect of the same compounds was studied in vitro in isolated papillary muscles and the IC50 values for inhibition of contraction determined. There was a statistically significant correlation between the IC50 values for binding and response (r = 0.79, P less than .005; rs = 0.78, P less than .005). Consistent with previous studies with several of these compounds, the response IC50 value for each compound was greater than the binding IC50 value. For most of the compounds, this difference was from one to two orders of magnitude. For three compounds, nitrendipine, nimodipine and nicardipine, this difference reached three orders of magnitude. These three dihydropyridine analogs share structural features that may determine their low myocardial potency and, at the same time, their high vascular smooth muscle potency. Elucidation of these structural features may be useful in determining which analogs will have the highest vascular smooth muscle selectivity.

Animals↗
PubMedDate not supplied

Decreased binding of verapamil to plasma proteins in patients with liver disease.

The binding of verapamil to proteins in plasma of patients with liver disease was studied using equilibrium dialysis. Compared with an age- and sex-matched control group, the fraction unbound of verapamil was significantly greater in patients with liver disease (p less than 0.01). The mean +/- SD fraction unbound in the patients was 0.16 +/- 0.05 compared with 0.099 +/- 0.015 in the control group (p less than 0.01). The higher fraction unbound in the liver disease patients appeared to be largely due to lower concentrations of binding proteins in plasma. A substantial effect of pH on the binding of verapamil to plasma proteins was observed. The increase in fraction unbound is consistent with previous findings of an increased apparent volume of distribution in patients with liver disease. Because of the pharmacokinetic characteristics of verapamil, the observed altered binding to plasma proteins would be expected to result in higher steady-state plasma concentrations of unbound drug after intravenous but not oral administration. For clinical monitoring, at any given total concentration, the unbound concentration would be approximately 60% higher in patients with liver disease. This study, together with previous studies in the literature, suggests that caution should be exercised in the administration of verapamil to patients with liver disease.

Adult↗
PubMedDate not supplied

The pharmacokinetics and pharmacodynamics of d- and dl-verapamil in rabbits.

The pharmacokinetics and pharmacodynamics of d- and dl-verapamil were studied in conscious rabbits in randomized cross-over fashion. Following a single intravenous dose, there was a biexponential decline in plasma concentration with time. No differences were observed in the pharmacokinetic properties of the compounds. The mean (+/- SD) clearances of d- and dl-verapamil were 0.13 +/- 0.03 and 0.12 +/- 0.05 L/min/kg, respectively. The mean (+/- SD) steady-state volume of distribution was 9.7 +/- 5.2 L/kg for d-verapamil and 8.1 +/- 4.1 L/kg for dl-verapamil. No difference was observed between the compounds in their binding to plasma proteins. The mean (+/- SD) half-life in plasma was 98.7 +/- 63.8 min for d-verapamil and 96.3 +/- 38.0 min for dl-verapamil. In contrast to the lack of stereoselective differences in the pharmacokinetic properties of verapamil, there were marked differences in the pharmacodynamics of d- and dl-verapamil. dl-Verapamil appeared to prolong the PR interval to a greater degree than did d-verapamil, consistent with the more potent calcium channel effects of the l-enantiomer. Similarly, dl-verapamil had more potent hypotensive effects compared with the d-enantiomer, which produced no effects on systemic arterial pressure. Chronotropic effects, judged to be caused by autonomic reflexes in response to the hypotensive effects of the compound, were also statistically greater for dl-verapamil than for d-verapamil. These results demonstrate stereo-selective pharmacodynamic effects in vivo of verapamil.

Animals↗