NUCLEOSIDE COMPOSITIONS CONTAINING PARACELLULAR ABSORPTION ENHANCERS
The present invention relates to a method for improving the absoφtion of nucleoside analogues such as (2R, cisH-amino-1-(2-hydroxymethyl-1 ,3- oxathiolan-5-yl) -(1H)- pyridmidin-2-one, also known as lamivudine, and 3'-azido- 3'-deoxythymidine, also known as zidovudine.
Lamivudine and zidovudine are reverse transcriptase inhibitors useful in the treatment of viral infections.
PCT patent application publication number WO 91/17159 describes the compound (2R, cis)-4-amino-1-(2-hydroxymethyl-1 ,3-oxathiolane-5-yl)-(1 H)- pyrimidin-2-one (also known as lamivudine) and its use in the treatment of HIV infections.
Lamivudine is the (-)-enantiomer of one of the compounds (BCH-189) described in EPA 0382526.
PCT patent application publication number WO92/11852, describes the use of BCH-189 and its individual enantiomers, including lamivudine, for the treatment of hepatitis B.
The use of lamivudine for the treatment of hepatitis C is described in International patent application no. PCT/EP96/03601.
The use of zidovudine in the treatment of AIDS is described in US Patent No. 4,724,232.
Nucleoside analogues such as lamivudine and zidovudine may be administered orally or intranasally and, following oral or intranasal administration, are absorbed paracellularly (i.e. through the tight junctions between cells of the intestinal mucosa). Enhancement of drug absorption is in general advantageous since this it enables lower doses to be effective (enhanced extent of absorption) and provides more rapid relief from symptoms (enhanced rate of absorption).
It has been reported that certain monosaccharides and amino acids stimulate cytoskeleton contraction to open up paracellular spaces to a sufficient size to pass molecules of a high molecular weight. Thus, Nellans (Nellans, H.N., Adv. Drug Delivery. 1991; 7:339-364) suggested that manipulation of the paracellular pathway could be used to enhance the oral delivery of small peptides and peptidomimetics. Some in vitro models have suggested that glucose may enhance paracellular absoφtion. However, Nellans (see above) failed to observe any positive effect on absoφtion using lumenal glucose in vivo suggesting that positive in vitro results may be offset in vivo by secretory water flow such that little or no increase in absorption is observed.
International patent application publication no. WO96/08238 discloses the use of paracellular absoφtion enhancers for enhancing the absoφtion of histamine H2 - antagonists.
Studies in intact rats with zidovudine (Fleisher, D. et al. Pharm. Res.. 7, no. 9, Suppl., S154, 1990) suggest that D-glucose may have an effect on the variability of zidovudine absoφtion.
A method of significantly enhancing the absorption of nucleoside analogues such as lamivudine and zidovudine following oral or intranasal administration has now been found.
Thus the present invention provides, in one aspect, the use of nucleoside analogues such as lamivudine and zidovudine or physiologically acceptable derivatives thereof and one or more paracellular absoφtion enhancers in the manufacture of medicaments for simultaneous, separate or sequential use for the treatment of viral infection characterised in that the paracellular absoφtion enhancer(s) significantly enhances the absoφtion of the nucleoside analogue.
In a further aspect, the invention provides a method of treatment of viral infection comprising orally or intranasally administering to a mammalian subject an effective amount of a pharmaceutical composition comprising a nucleoside analogue such as lamivudine or zidovudine or a physiologically acceptable derivative thereof and one or more paracellular absorption enhancers, wherein
the paracellular absoφtion enhancer significantly enhances the absorption of the nucleoside analogue.
As used herein "physiologically acceptable derivative" means a physiologically acceptable salt, ester or salt of such ester.
The term "paracellular absorption enhancer" as used herein encompasses any compound which enhances paracellular absoφtion. For example, the paracellular absoφtion enhancers are those which occur naturally in nutrients. Paracellular absoφtion enhancers include carbohydrates such as monosaccharides, e.g. glucose, galactose, mannose, 3-0-methyl glucose, xylose, ribose, arabinose, ribulose, fructose and sorbose. The monosaccharides may be employed in either their D- or L- forms. Where the monosaccharide is naturally occurring, the naturally occurring form is preferred.
Preferred paracellular absoφtion enhancers include glucose, e.g. D-glucose. A further preferred group of paracellular absoφtion enhancers includes galactose, e.g. D-galactose, mannose, e.g. D-mannose, 3-0-methyl glucose, e.g. 3-0- methyl D-glucose, xylose, e.g. D-xylose.
It will be appreciated that the paracellular absorption enhancer(s) employed in the instant invention will be of the reversible type i.e. one whose absorption enhancement effect rapidly diminishes when it is no longer present at the site of action. All of the paracellular absoφtion enhancers specifically mentioned above are of the reversible type.
The paracellular absorption enhancers may be used alone or in combination.
Nucleoside analogues may be used alone or in combination in the compositions, methods and uses of the invention. In particular, lamivudine and zidovudine may be used in combination.
It will be appreciated that reference to treatment is intended to include prophylaxis as well as the alleviation of established symptoms.
As mentioned hereinbefore, paracellular absorption enhancers have been found to significantly enhance the absoφtion of nucleoside analogues such as lamivudine and zidovudine following oral or intranasal administration. Suφrisingly, both the extent and rate of absoφtion are enhanced.
Thus, according to a further aspect, the present invention provides a method of significantly enhancing the rate of absorption of a nucleoside analogue such as lamivudine and zidovudine following oral or intranasal administration by simultaneous, separate or sequential administration of a nucleoside analogue such as lamivudine and zidovudine with one or more paracellular absorption enhancers.
Nucleoside analogues such as lamivudine and zidovudine or physiologically acceptable derivatives thereof and one or more paracellular absorption enhancers may be co-administered in the form of separate pharmaceutical compositions for simultaneous and/or sequential use. Preferably, a nucleoside analogue such as lamivudine or zidovudine and paracellular absorption enhancer(s) are administered as a single pharmaceutical composition for oral or intranasal use comprising effective amounts of the active ingredient.
Thus, according to a further aspect, the invention provides a pharmaceutical composition for oral use comprising a nucleoside analogue such as lamivudine or zidovudine or a physiologically acceptable derivative thereof and one or more paracellular absoφtion enhancers.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); a glidant (e.g. corn starch, colloidal silicon dioxide), lubricants (e.g. magnesium stearate, talc sodium sterol fumarate or silica); disintegrants (e.g. potato starch or sodium starch glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for
example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl- p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.
For intra-nasal administration the compounds of the invention may be used as a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one more more dispersing agents, soiubilising agents or suspending agents. Liquid sprays are conveniently delivered from pressurised packs. Dispersible powder formulations typically comprise a powder mix of the active ingredient(s) and a suitable powder base such as lactose or starch.
Particularly suitable pharmaceutical compositions according to the present invention are tablets and liquid syrups or suspensions for oral administration
Suitable methods of formulation are known in the art and include those methods described in WO 91/17159 and US Patent No. 4,724,232, which are incoφorated herein by reference.
The paracellular absorption enhancer(s) may be incorporated into the above- mentioned formulations according to conventional procedures.
Nucleoside analogues and paracellular absorption enhancer(s) may, if desired, be administered in combination with one or more other therapeutic agents and formulated for administration by any convenient route in a conventional manner. Appropriate doses will be readily appreciated by those skilled in the art.
The ratio of nucleoside analogue to paracellular absorption enhancer(s) used in the method or compositions according to the invention may be in the range of
1:1 to 1:1000 (by weight), such as 1:1 to 1:50 or 1:150 (by weight), for example 1 : 5 (by weight).
The amount of paracellular absoφtion enhancer used in the oral formulations according to the instant invention is in the range of 10 to 3000mg, e.g. 20 to 1000mg, per dosage unit.
The amount of the nucleoside analogue used in the oral formulations according to the instant invention is preferably in the range of 5 to 800mg per dosage unit. For example, 20mg to 700mg per dosage unit.
The unit dose (for example contained in one tablet according to the invention) may be administered for example, 1 to 4 times a day.
The following are illustrations of non-limiting examples of pharmaceutical compositions according to the invention.
Example 1
Tablets for oral administration
Lamivudine or zidovudine 150mg
D-glucose 150mg microcrystalline cellulose 80mg magnesium stearate 5mg sod iun starch glycolate 15mg
Example 2
Capsule
The formulation of Example is filled into two part gelatine capsules.
The formulation of Example is filled into two part gelatine capsules. Solution for oral administration
Lamivudine or zidovudine 10mg
D-glucose 10OOmg methylparaben 1.2mg propylparaben 0.15mg edetate disodium 0.1mg ethanol 50.8mg propyiene glycol 20mg flavour 1.4mg citric acid . 1.2mg purified water to 1ml
The above formulations are prepared by admixture of the ingredients using conventional pharmaceutical techniques.
Biological Data
Enhancement of the extent and rate of transport of lamivudine and zidovudine by D-glucose was measured in Caco-2 cells using the method set out below.
The results are presented in Table 1.
Caco-2 cells, originating from a human colorectal carcinoma, were obtained from Porton Down (ECACC No: 86010202). Cells were allowed to grow and differentiate on polycarbonate membrane culture plate inserts (Transwells™ 12mm diameter, 0.4μm pore size; Costar UK Ltd, High Wycombe, Bucks). The cells were maintained in minimum essential medium (Eagle's) containing 10% bovine calf serum (Gibco BRL), 1% non-essential amino acids, penicillin (100U/ml), streptomycin (10μg/ml) and L-glutamine (2mM). All tissue culture
media (except where stated) were obtained from Hycione Europe Ltd. The medium was changed three times a week.
The medium used for carrying out the transport studies was a glucose buffer formula based on Kreb's bicarbonate solution. Termed ORS buffer, this contained a high concentration of glucose compared to the control buffer. Transport rates were measured on compounds in both the control and ORS buffers to determine the effect of a high concentration of glucose. The pH of both buffers was approximately 6.4.
The constituents of each buffer are detailed in the table below:
A solution of each compound at a concentration of 1 mM was prepared in both the control and ORS buffers. Each solution was spiked with a small amount of the respective radiolabelled compound to facilitate detection.
Cell transport studies were performed at 37°C in a water bath with gentle shaking. The cells used had been grown for a period of 21 to 26 days. The cells were acclimatised for 30 to 60 minutes in control buffer prior to the start of the experiment. During this time, the trans-epithelial electrical resistance (TEER) across the cells was measured with a volt-ohmmeter. This is used as a measure of tight junction integrity. Transport rates were measured in the apical (AP) to basolateral (BL) direction in both control and ORS buffer, and also in the BL to AP direction. Studies were initiated by adding the correct buffer/ compound concentration as presented below:
A B C D
Apical side Control Buffer ORS Buffer Control Buffer Control Buffer + compound + compound
Basolateral Control Buffer Control Buffer Control Buffer ORS Buffer side + compound + compound
At 5, 10, 15, 20, 30, 60, 90 and 120 minutes after the start of the experiment, an aliquot from the receptor compartment was removed and added to scintillation fluid prior to liquid scintillation counting. The volume removed was replaced with an equal volume of control buffer. At the end of the experiment the TEER was re-measured.
Aliquots of the receptor compartment at the end of the experiment were analysed by radio-HPLC and compared to an aliquot of the donor compartment at the begining of the experiment to ensure that the compound was transported intact. A further aliquot of the compound in buffer was kept at 37°C for the duration of the experiment and analysed to ensure that the radio-purity of the compound did not deteriorate at this temperature.
TABLE 1
Effect of D-Glucose on Transport of Nucleoside Analogues across Caco-2 cells
Compound Rate of Rate of nmols nmols Rate of nmols transport transport transported transported transport transported
30-120 30-120 at 2h at 2h 30-120 at 2h mins mins mins
Control ORS Control ORS ORS as % ORS as % Control Control lamivudine 0.34 1.04 38 166 306% 437%
zidovudine 0.99 1.46 122 169 147% 139%