William J. Irwin

Biodegradable poly(L-lactic acid) matrices for the sustained delivery of antisense oligonucleotides

Abstract Antisense oligonucleotides have emerged as exciting novel therapeutic agents which can inhibit gene expression in a sequence-specific manner and are currently undergoing clinical trials evaluation in the treatment of cancer and viral diseases. The therapeutic application of these molecules is limited by their poor biological stability and rapid in vivo elimination kinetics which necessitates frequent administration of oligonucleotides for sustained efficacy. The potential use of a sustained-release biodegradable delivery system, that would protect oligonucleotides from degradation by nucleases whilst delivering the nucleic acid in a controlled or sustained manner to the site of action, may circumvent these problems. In this study, we report on the biological stability, hybridization potential and in vitro release kinetics of antisense phosphodiester (D-oligos) and phosphorothioate (S-oligos) oligonucleotides entrapped within biodegradable poly( l -lactic acid) (PLA) matrices. The in vitro release profiles of 5′-end radiolabelled oligonucleotides entrapped within solvent-cast polymer film matrices (100±10 μ m thickness) were monitored over a period of at least 28 days in either serum, citrate buffer (pH 5.5) or phosphate buffer (pH 7.4). The release profiles over 28 days suggested that the entrapped oligonucleotide was released biphasically from the polymer films, characterized by a rapid burst release during the first 48 h followed by a more sustained release. Oligonucleotide release from PLA matrices was dependent on oligomer chemistry (S-oligos were released more slowly than D-oligos) and on oligomer length (a 20-mer S-oligo was released more slowly than a 7-mer). During the release experiments, little or no degradation of the polymer matrices was observed by SEM or by DSC methods. Oligonucleotide release could be described by the mathematical model for drug release from a solution within a monolithic polymer slab. In all cases, the polymer-entrapped oligonucleotides were resistant to degradation from serum nucleases over the entire study period whereas free phosphodiester oligonucleotides were completely degraded within 1 h. Gel mobility shift analyses and duplex melting point determinations suggested that the hybridization capability of antisense oligonucleotides released from the polylactide matrices was unaffected by the solvent-casting procedure for preparing sustained release polymer devices. These results suggest that biodegradable polymer matrices may be suitable delivery systems for the sustained administration of antisense oligonucleotides.

Drug-Delivery by Ion-Exchange: Part IIII: Interaction of Ester Pro-Drugs of Propranolol with Cationic Exchange Resins

AbstractThe interaction of a series of O-n-acyl propranolol prodrugs with strong cation exchange resins is reported and various variables which control loading and release profiles have been investigated. pH has little practical effect on the loading efficiency under the conditions used but standardisation of experimental details for the measurement of in vitro liberation profiles must be undertaken. In particular, stirring speeds of 200-300 rpm were necessary to ensure the independence of release profile and agitation. Additionally, the dissolution medium may not provide sink conditions throughout the full dissolution process if pH variation does not take into account solubility differences between analogues. Ionic strength also influences release rates and attempts to examine the effects of pH must also control this variable. The proportion of cross-linking agent in the resin plays an important role. Increasing the cross-linking delays considerably the release of drug from the matrix and is a useful par...

The Effect of Selected Water-Soluble Excipients on the Dissolution of Paracetamol and Ibuprofen

The purpose of this investigation was to study the dissolution behavior of paracetamol and ibuprofen in the presence of a range of selected potential excipients. First, a pH-solubility profile was generated for both drugs, and the effect of changing hydrodynamic conditions on the intrinsic dissolution rate was investigated. It was established that both drugs dissolved according to the diffusion-layer model. Paracetamol solubility (approximately 20.3 mg mL− 1) did not vary from pH 1.2–8.0, corresponding to the in vivo range in the gastrointestinal tract. Ibuprofen had an intrinsic solubility of approximately 0.06 mg mL− 1, and pKa was calculated as 4.4. Second, the effects of selected potential excipients (lactose, potassium bicarbonate, sodium bicarbonate, sodium chloride, and tartaric acid) were evaluated by measuring the effect of the inclusion of each additive in the dissolution medium on drug solubility, drug intrinsic dissolution rate, and solution viscosity. The results were evaluated using the diffusion-layer model, and it was determined that for paracetamol, the collected data fitted the model for all the excipients studied. For ibuprofen, it was found that there were differences between the excipients that raised the solution pH above the pKa to those that did not. For the excipients raising the pH above the pKa, the effect on intrinsic dissolution rate was not as high as that expected from the change in drug solubility. It was postulated that this might be due to lack of penetration of the excipient into the drug boundary layer microenvironment. Formulators may calculate the effect of adding an excipient based on solubility increases but may not find the dissolution rate improvement expected.

Development of a Sustained-Release Biodegradable Polymer Delivery System for Site-Specific Delivery of Oligonucleotides: Characterization of P(LA-GA) Copolymer Microspheres In Vitro

Development of a Sustained-Release Biodegradable Polymer Delivery System for Site-Specific Delivery of Oligonucleotides: Characterization of P(LA-GA) Copolymer Microspheres In Vitro Antisense oligodeoxynucleotides (ODNs) can selectively inhibit individual gene expression provided they gain access to and remain stable at the target site for a sufficient period of time. Biodegradable sustained-release delivery systems may facilitate site-specific delivery and also prevent degradation of ODNs by nucleases whilst delivering the nucleic acid in a controlled manner to the desired site of action. In this study, we have characterized biodegradable poly (lactide-co-glycolide) (P(LA-GA)) 50:50 microspheres for the potential delivery of antisense oligonucleotides in vivo. Phosphodiester (PO) oligonucleotides complementary to either c-myc proto-oncogene or the tat gene in HIV-RNA were adequately incorporated within P(LA-GA) microspheres with entrapment efficiencies up to 60% depending on particles size. In vitro release profiles of antisense nucleic acids from 10-20 microm size microspheres over 56 days in physiological buffer were triphasic. Profiles were characterised by an initial burst effect during the first 48 hours (phase 1) of release followed by a more sustained release (phase 2) with an additional increased release (phase 3) being observed after 25 days which corresponded with bulk degradation of the copolymer matrix. The release profiles were influenced by microsphere size, copolymer molecular weight, ODN loading, ODN length and by the pH of release medium used. The serum stability of PO ODNs was significantly improved when entrapped within P(LA-GA) microspheres and the hybridization capability, as assessed by duplex melting (Tm) measurements, of released ODN was not impaired by the double-emulsion microsphere fabrication procedure used. Thus, P(LA-GA) microspheres appear to be promising candidates for improving site-specific delivery profiles for ODNs and are worthy of further evaluation in vivo.

Low Intensity Ultrasound as a Probe to Elucidate the Relative Follicular Contribution to Total Transdermal Absorption

AbstractPurpose. To investigate the effect of ultrasound on the histological integrity and permeability properties of whole rat skin in vitro. Methods. A defined, field-free source of ultrasound was used to irradiate excised rat skin prior to in vitro transport studies in Franz-type cells using sucrose, mannitol, hydrocortisone, 5-fluorouracil and aminopyrine. Results. High intensity ultrasound irradiation (1 to 2 W cm−2) irreversibly damaged cutaneous structures and increased the percutaneous transport rate of permeants. In contrast, skin integrity was largely maintained with low intensity ultrasound (0.1 to 1 W cm−2) which merely discharged sebum from the sebaceous glands so as to fill much of the hair follicle shafts. This effect caused the transfollicular absorption pathway to be blocked for hydrophilic molecules that penetrate via this route and reduced the transport rate significantly. Conclusions. This phenomenon may be used as a probe to elucidate the relative follicular contribution to total penetration for hydrophilic permeants. It was demonstrated that the shunt pathway was responsible for virtually all mannitol and sucrose penetration, perhaps half of hydrocortisone transport but negligible aminopyrine and 5-fluorouracil penetration.

Drug delivery via active transport at the blood-brain barrier : affinity of a prodrug of phosphonoformate for the large amino acid transporter

Abstract Due to its hydrophilic nature, the antiviral agent phosphonoformate (PFA) is excluded from the CNS by the blood-brain barrier (BBB). Lipophilic triesters of PFA, designed to penetrate the BBB have been found to be unsuitable as prodrugs due to their rapid and complicated hydrolysis. Hydrophilic drugs, such as l -dopa, are known to cross the blood-brain barrier by means of an active amino acid transporter. Thus, the possibility that a PFA-amino acid conjugate may be actively transported at the BBB was investigated. A PFA- l -tyrosine conjugate [sodium 4-(2′-carboxyl-2′-aminoethyl)phenyl methoxycarbonylphosphonate] was synthesised and characterised. Active amino acid transport was studied in vitro using monolayers of porcine brain microvessel endothelial cells. Confluent monolayers were obtained after 4–5 days in culture, and alkaline phosphatase activity and the presence of Factor VIII antigen were demonstrated histochemically. The transport of l -[3H]tyrosine was shown to be temperature- and concentration-dependent and transport constants were calculated to be Km = 0.149 mM and Vmax = 3.07 nmol/h per insert by non-linear regression. l -Dopa and other large amino acids were found to inhibit the transport of l -[3H]tyrosine, consistent with their transport by the amino acid transporter in vivo. The PFA- l -tyrosine conjugate, which was stable under the experimental conditions, also inhibited l -[3H]tyrosine transport indicating that it may be a substrate for active transport at the BBB.

Proline uptake by monolayers of human intestinal absorptive (Caco-2) cells in vitro

Monolayers of the Caco-2 human intestinal cell line exhibit active and passive uptake systems for the imino acid L-proline. The active transport component is saturable and it is responsible for about two thirds of the observed flux over the nanomolar concentration range, at 37 degrees C and pH 7.4. In contrast to L-phenylalanine, specific L-proline uptake has a high degree of sodium dependency and the efficiency of the carrier system is significantly reduced when protein synthesis (cycloheximide), Na+/K(+)-ATPase (ouabain) or cellular metabolism (sodium azide) are inhibited. The expression of the L-proline carrier by Caco-2 cells was under some degree of nutritional control. Glucose deficiency, over the time scale of the experiment, had no effect. The temperature-dependence of the specific uptake process followed the Arrhenius model with an apparent activation energy of 93.5 kJ nmol-1. This pathway also displayed Michaelis-Menten concentration-dependence with a Ksdm of 5.28 mM and a maximal transport flux (Jsdmax) of 835 pmol min-1 (10(6) cells)-1. Although the passive component was unchanged, the pH of the donor phase exerted a profound effect on the active carrier component. Within the physiological pH range a local maximum efficiency was found at pH 7.4 but dramatic increases were noted as pH 5.0 was approached. In competition studies, with 100-fold excess of a second amino acid, strong inhibition of uptake was found with alpha-aminoisobutyric acid, L-alanine and L-serine whereas moderate inhibition was observed with glycine, D-proline and gamma-aminoisobutyric acid. Aromatic and branched amino acids showed weak (L-valine) or no interaction (L-phenylalanine, L-leucine) with the carrier system. These data indicate that the carrier system for the uptake of L-proline has many features in common with the A system for amino acid transport.

Ionisation and the effect of absorption enhancers on transport of salicylic acid through silastic rubber and human skin

PURPOSE to investigate if salicylic acid (SA)-permeation through excised human skin (HS) and silastic rubber (SR) conforms to the pH-hypothesis and to assess the influence of a range of absorption enhancers on the transport of SA with and without a transmembrane pH-gradient. METHODS Franz cells were used to study SA permeation from solutions and saturated suspensions. McIlvaine buffers were used to maintain transmembrane pH-gradients. Membrane pretreatment was used to study the action of absorption enhancers. RESULTS the flux of SA from solutions was dependent upon the vehicle pH and permeant concentration was directly related to the degree of ionisation of the solute. Flux from suspensions was independent of pH, since the level of unionised drug, the predominant diffusing species, was maintained at the intrinsic saturated solubility at all pH values. The observed SA flux enhancement across human skin without a transmembrane pH-gradient was not significantly different from the enhancement with a pH-gradient for all of the absorption enhances used, except for dodecylamine. CONCLUSIONS the results showed that SA permeation conformed to the p H-partition hypothesis. The evidence from absorption-enhancer pretreatment demonstrated that, under certain conditions, the transdermal penetration enhancement of a number of topical enhancing compounds, including Azone and oleic acid can be explained without recourse to ion-pair phenomena.

A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 2: affinities of ACE inhibitors.

PURPOSE To assess the affinities of a series of ACE inhibitors for the di/tri/oligopeptide transport system (DTS) using a rapid in vitro system. METHODS Monolayers of Caco-2 cells were cultured in plastic wells for 7-9 days and the uptake of Gly-[3H]L-Pro was used as an affinity probe. Gly-[3H]L-Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [3H]L-Pro produced by degradation of the probe, was incubated with the test compound (usually 1 mM) at pH 6 for 3-mins. The uptake of radiolabel was determined by liquid scintillation counting. RESULTS A 2-dimensional six-domain model of the transporter based on the structure of a phosphinate ACE inhibitor (SQ-29852) was constructed to facilitate interpretation of the competitor affinities. The SQ-29852 molecule was divided into six binding domains (A-F) based on functional groups within these regions and the effects of structural variation in four of these domains (A, C-E) were explored. A series of dipeptide-like compounds varying within specific domains were selected from a large number of commercially available ACE inhibitors and SQ-29852 analogues. Domain A had a preference for an uncharged group, with bulky hydrophobic groups reducing affinity. Domain C exhibited a preference for a positive charge over a neutral function, with the space this functional group occupies contributing to affinity. Domain D favoured lipophilic residues and domain E retained activity when the carboxylic acid was esterified. CONCLUSION The test system is able to reveal structure-activity relationships of peptidomimetic agents and may well serve as a design tool to optimise affinity for the DTS.

Phonophoresis - is it a reality?

Abstract Phonophoresis - the application of ultrasound to enhance percutaneous drug delivery - has been administered by physiotherapists for over 30 years. However, since the treatment has been conducted on a highly subjective and non-quantitative basis, no clear consensus exists on the effectiveness of the technique nor on the nature of the phonophoretic mechanism. Ultrasonic energy can perturb mammalian tissue via its heating, radiation pressure, cavitation and acoustic microstreaming effects and each of these is discussed in turn in relation to topical drug delivery. Evidence from the literature is reviewed from the separate perspectives of in vitro research, animal studies and human volunteer trials. It may be concluded that phonophoresis is indeed a reality for certain molecules under certain conditions and that ultrasonic heating is its main though not exclusive mechanism of action. However, at present the therapeutic value of the technique is still under question.