Polymeric Based Complex
The present invention relates to novel compounds comprising a lipid metabolite for maintaining cellular viability, and to a method of administering the compounds to a cell.
Breeding programmes of animals usually involve the use of artificial insemination. The viability of spermatozoa can decrease greatly over time during storage. After five days it is common for the viability of the spermatozoa to decrease to such an extent to render it commercially unviable. The motility of the spermatozoa is also greatly affected by storage. It is therefore common for spermatozoa to be discarded after a short period as it is considered to be ineffective for artificial insemination after that time. This clearly has a significant affect on the cost of conducting artificial insemination.
It has previously been recognised that highly unsaturated long chain fatty acids and/or lipid moieties containing such acids, play a specific role in the maintenance and promotion of a wide range of essential metabolical and physiological functions of animal cells. To enable maximisation of these functions, an appropriate level of antioxidant capacity is necessary. Under normal circumstances however, we have found that vitamin-E does not enter the spermatozoa cells in sufficient quantities to maintain viability.
The present invention addresses the problems inherent to delivery of lipids to cells under aqueous environments, through the concept and application of polymeric based complexes providing . the delivery to cells of linked permutations/combinations of identified lipid metabolites.
According to a first aspect of the present invention there is provided a polymeric based complex comprising a comonomer, a saccharide residue and at least one lipid metabolite for retaining cellular viability.
The saccharide residue may be any carbohydrate residue, suitably a saccharide group, including the D- or L- enantiomer of any mono-, di~, tri-, oligo- or poly-saccharide, wherein each saccharide unit may be the alpha- or beta-anomer. Preferably the saccharide residue comprises one or more glycosyl
groups, more preferably the saccharide residue is beta-D-galactose .
The lipid metabolite may be a fatty acid, (particularly a partially or fully unsaturated long chain fatty acid) , a lipid moiety comprising such an acid, a vitamin, an antioxidant, a peptide, (including an oligopeptide) , an organoselenium compound, a stimulant (particularly a selenium based compound or caffeine) , or any precursor of these that may. form the lipid metabolite in vitro or in vivo or a combination thereof, suitably in the form of a chemically linked complex. Preferably the lipid metabolite is an anti-oxidant, more preferably alpha-tocopherol (vitamin E) optionally together with a stimulant such as- caffeine.
The comonomer may suitably act as a linking group to link the saccharide residue and the lipid metabolite together. Suitaby the comonomer comprises a carbon-carbon double bond or an ester group, and preferably comprises both. The comonomer is suitably an acrylate, a methacrylate, a styrene, an acrylamide, an allyl, a crotonate, an itaconate, a vinyl, a cyclic ketene acetal, a cyclic ketene aminal or is formed from a combination of any of these. Preferably the comonomer is an acrylate or methacrylate.
The saccharide residue and comonomer may be combined to form a saccharide comonomer. The
functional group of the comonomer can be reacted together with the saccharide in order to link the two. Once linked the comonomer becomes functionalised with the saccharide and hence becomes a saccharide comonomer. Many examples of this exist in the scientific literature.
The lipid metabolite and comonomer may be combined to form a lipid metabolite comonomer. As in the case of saccharides, lipid metabolites can be modified such that they possess comonomer functionality. A lipid metabolite and a functional comonomer can be coupled together in order to form a lipid metabolite comonomer. Many examples can be found in the scientific literature.
The polymeric based complex may also comprise a solubilising group, wherein the solubilising group improves the solubility of the polymeric based complex in a liquid media, suitably the solubilising group is hydrophilic or lipophilic. The solubilising group suitably comprises an amine or ammonia group. The solubilising group may be an ester (suitably an alkylester or a hydroxyethylester, preferably it is derived from N,N' -dialkylaminoethyl ester), a substituted aromatic group, an amide (particularly a primary, secondary or tertiary amide) , a trialkylammonium salt, a poly (ethylene glycol), a poly (ethylene oxide), a poly (propylene glycol), a poly (propylene oxide) , a poly (lactide) , a poly (glycolide) , a poly (lactide-co-glycolide) , a poly (eta-
caprolactone) , an oligopeptide, a polypeptide a poly (amino acid), or a combination thereof.
The polymer may be formed by any accepted method of performing addition polymerisation, including initiation by radicals, anions, cations, organometallic species, enzymes and redox methods. In the present embodiment, the polymer is preferably prepared by addition polymerisation initiated by free radicals. ■ Preferably the polymeric based complex comprises at . • least two lipid metabolites, more preferably three to five lipid metabolites.
Preferably the polymeric based complex is lipid or aqueous soluble.
According to a further aspect of the present invention there is provided a composition comprising the polymeric based complex and at least one pharmaceutically acceptable excipient.
According to a further aspect of the present invention there is provided a method of administering one or more lipid metabolites to a cell, said method comprising the step of administering a composition comprising a polymeric based complex as described above to the cell for maintaining cellular viability.
Preferably the polymeric based complex enters the cell.
The present invention describes a polymeric based complex which acts as a single entity carrier system for multiple components (the lipid metabolite and the saccharide residue) and exhibits the flexibility of a controlled and directed delivery of lipid metabolites to cells under a variety of conditions. Through the delivery of such combinations, synergistic effects may be exerted between the supplied metabolites on their individual cellular availability and activity, to maximally promote cellular function.
Polymeric based complexes comprising alpha- tocopherol chemically bound through a phosphate linkage to fatty acid(s) or other lipid metabolites are especially preferred and the conjugation of these active groups provides an opportunity for metabolite presentation and subsequent cellular uptake. In the past however, their apolarity has presented obvious difficulties for cells maintained in basically aqueous environments. Such polymeric based complexes can be prepared by polymerisation of polymerisable derivatives of carbohydrate targeting residues, polyunsaturated fatty acids and/or their specific lipid complexes, appropriate antioxidants, other lipophilic moieties, etc. This approach leads to soluble polymer molecules that contain such beneficial lipid metabolites, which are easily accessible to
the cell for subsequent uptake and metabolisation, and maintain cellular viability.
The present invention utilises selective chemistry to introduce a saccharide residue to the polymeric based complex to exploit the active-transport mechanisms inherent to cells, through specific recognition of the saccharide residue by cells. This precise strategy will afford absolute control over the nature and levels of delivery of lipid metabolites without' the potential for the erroneous • delivery of undesirable materials and can be fine- tuned in terms of particle size, spatial arrangement, metabolite range and concentration to suit differing cell types and their environmental situations.
The .present invention clearly demonstrates the ability to prepare and practically employ the multi-component polymeric based complexes as cellular delivery systems for the incorporation and subsequent metabolism of lipid metabolites to maintain cellular viability. The presence of chemical functional groups on the polymer allows covalent attachment of various agents while the presence of at least one saccharide residue facilitates a targeted delivery to the cell. According to one aspect of the present invention the lipid metabolite (s) are linked to the polymer through weak, enzymatically labile ester bonds. Whilst the Applicant does not wish to be bound by theory it is believed that once inside the cell the
lipid metabolites can be released by hydrolysis of their weak enzymatically labile ester bonds to the polymer.
The cell is suitably a male or female gamete, suitably a fish, bird or mammal gamete, particular mention may be made of human, pig, cattle, goat, sheep and poultry gametes .
Preferably the gametes are stored under fresh, cryopreservation and verification storage conditions.
Suitably the method comprises administering the composition to spermatozoa within 1 hour of ejaculation causing the spermatozoa to have a mean motility rate of more than 50%, five days after ejaculation.
Suitably the method comprises administering the composition to spermatozoa within 1 hour of ejaculation causing the spermatozoa to have a mean viability rate of more than 70%, eight days after ejaculation.
Suitably the method comprises administering the composition to spermatozoa within 1 hour of ejaculation causing the longevity of the spermatozoa to increase by 90% or more, preferably by 100% or more.
The present invention provides the first
demonstrative exploitation of the receptor-mediated endocytotic active transport mechanism to cells through the surface recognition of a polymeric based complex bearing a saccharide residue (suitably a carbohydrate residue, preferably beta- D-galactose) . - This allows the uptake and delivery to the spermatozoa of specifically polymer bound lipid metabolites and the prevention of any uptake of non-desirable materials. • Absolute control over the nature and level of beneficial metabolite delivery is therefore afforded.
The highly positive effects observed in evaluating spermatozoa function and viability show the present invention has practical application in the field of modern breeding of pigs and other intensive commercial animal systems and will have positive financial and time implications. These positive results also illustrate the application of the present ■ invention to human fertility treatment . The methodology also embraces its application to the controlled promotion of the delivery and uptake of specific nutrients and metabolites for- the enhancement of oocyte maturation and early fertilised cell survival in situations of in vitro fertilisation.
Preferably the method comprises the step of promoting the fertilisation of an egg in-vi tro by administering the polymeric based complex to the spermatozoa prior to exposing the spermatozoa to the egg.
Preferably the method comprises the step of promoting the viability of sex sorted cells to be fertilised by administering the polymeric based complex to the spermatozoa, prior to and after separating the spermatozoa with an X-chromosome from the spermatozoa with a Y-chromosome .
Preparation of the multi-component polymeric based complex involves modification and combination of existing methodologies for free radical polymerisation. - .According to one method of production, monomer units may be specifically designed to introduce, in a highly controlled manner, sugar targeting residues and alpha- tocopherol to form a combined polymer package for . specific cellular delivery. This technique presents scope for the preparation of a wide range ' of multi-component polymers with different chemical ■ properties i.e. hydrophilic or hydrophobic, charged . or neutral, biodegradable or .inert, presenting enormous scope for the delivery of necessary ■ • metabolites required to promote cell metabolism, viability and functionality.
The present invention will now be described by way of example only with reference to the accompanying drawings.
A polymer product incorporating beta-D-galactose (cellular cognitive compound) and alpha-tocopherol (lipophilic antioxidant) was prepared as described below.
Initial attempts of production focused on the free radical copolymerisation of vinyl monomers derived from alpha-tocopherol and beta-D-galactose pentaacetate, which were prepared according to procedures described in the literature (Schmidt, R. Angew. Chem . 1986, 25, 212-235; Singh, N, Schmidt, R. J. Carbohydr. Chem . 1989, 8, 199-216; Ortiz, C, Vazquez, B., San Roman, J. Polymer 1998, 39, 4107- 4114) . However, no combinations of these monomers in any ratio were found to lead to the production of polymeric material . Due to the crowded nature of each of these molecules, it was suspected that high steric demands were preventing copolymerisation and so a further smaller monomer molecule was added to reduce steric crowding and thus promote polymerisation.
The following examples describe the successful production of terpoly ers containing beta-D- galactose, alpha-tocopherol and a third vinyl monomer compound (either 2 -hydroxyethyl acrylate, 2 -hydroxyethyl methacrylate, 2- (dimethylamino) ethyl acrylate) or 2- (dimethylamino) ethyl methacrylate).
Example 1
A solution of 2- (2 ' , 3 ' , 4' , 6' -tetra-O-acetyl-beta-D- galactosyl) ethyl methacrylate (360mg, 0.78mmol), alpha-tocopheryl methacrylate (lOOmg, 0.20mmol), 2- hydroxyethyl methacrylate (43mg, 0.33mmol) and AIBN (3.4mg, 1.5wt% w.r.t. monomers) in anhydrous DMF (2.2ml) was prepared. The solution was flushed
with nitrogen and then heated at 50oC under an atmosphere of nitrogen with continuous stirring for 24 hours. Subsequently the solvents were removed under vacuum and a 1H NMR spectrum of the crude material was obtained. The crude mixture was then purified by precipitation of the polymer from acetone into petroleum ether, collected by filtration and dried in vacuo . The polymer product was found to be insoluble in water. The polymer product was designated CF3.
Example 2
The beta-D-galactosyl residues in the polymer prepared as described in Example 1 were deacetylated as follows: the polymer (150mg) was dissolved in a mixture of anhydrous methanol (5ml) and anhydrous dichloromethane (3ml) and a solution of sodium methoxide in methanol- (0.29ml, 0.0IM) was added. The solution was stirred for approximately 1 hour 25 minutes at ambient temperature before adding DOWNEX 8W50 ion exchange ' resin. The solution was then filtered, the solvents removed under vacuum and the product recovered by freeze frying. This yielded 100 mg of product, which was found to be water soluble. The product was designated CF3 (modified) .
Example 3
A solution of 2- (beta-D-galactosyl) ethyl methacrylate (60mg, 0.20mmol), alpha-tocopheryl
methacrylate (25mg, 0.05mmol), 2 -hydroxyethyl methacrylate (llmg, O.Oδmmol) and AIBN (0.8mg, 1.5wt% w.r.t. monomers) in acetone (1ml) was treated as in Example 1 to yield a polymer product as a precipitate. This was collected by filtration and was found to be sparingly soluble in water. The product was designated CF8. A 1H NMR spectrum • (D20, 200 MHz) indicated that the product did not contain any alpha-tocopheryl residues.
Example 4
A solution of 2- (2 ' , 3 ' , 4 ' , 6' -tetra-O-acetyl-beta-D- galactosyl) ethyl acrylate (60mg, 0.13mmol), alpha- tocopheryl acrylate (15mg, 0.03mmol), 2- (dimethylamino) ethyl acrylate (225mg, 1.57mmol) and AIBN (4.5mg, 1.5wt% w.r.t. monomers) in anhydrous DMF (2.2ml) was treated as in Example 1 to yield a polymer product. The polymer was then purified by precipitation from THF into petroleum ether, filtered and dried in vacuo. The polymer was found to be soluble in water. This product was designated CF21.
Example 5
The beta-D-galactosyl residues in the polymer prepared as described in Example 4 were deacetylated as described in Example 2. The product obtained was designated CF21 (modified) .
Example 6
A solution of 2- (beta-D-galactosyl) ethyl acrylate (150mg, 0.539mmol), alpha-tocopheryl acrylate (150mg, 0.309mmol), 2- (dimethylamino) ethyl acrylate (450mg, 3.14mmol and AIBN (11.3mg, 1.5wt% w.r.t. monomers) in anhydrous DMF (2.2ml) was treated as in Example 1 to yield a polymer product . The polymer was then purified by precipitation from acetone into petroleum ether, filtered and dried in vacuo. The polymer was found to be soluble in water. This product was designated CF34.
Figure 1 shows the 1H NMR spectrum of polymer CF3.
Figure 2 shows the 1H NMR spectrum of polymer CF34.
Figure 3 shows a liquid chromatographic separation of polymer bound and free alpha-tocopherol .
Figure 4 compares the ability of a spermatozoa cell to take up polymer bound alpha-tocopherol and subsequent release of free-alpha tocopherol.
Figure 5 shows the levels of free alpha-tocopherol levels in spermatozoa as a function of polymer inclusion.
Figure 6 shows the levels during storage of free alpha-tocopherol in spermatozoa upon introduction of CF28 polymer compared to the levels upon introduction of a control.
Figure 7 shows the levels during storage of free alpha-tocopherol in spermatozoa upon introduction of CF34 polymer compared to the levels upon introduction of a control .
Figure 8a shows the concentration of malondialdehyde in spermatozoa after incubation for 2 hours at 37°C in the presence of polymer CF34 compared to the concentration after incubation for 2 hours at 37°C in the presence of a control.
Figure 8b shows the concentration of malondialdehyde in spermatozoa after incubation for three days at 18°C in the presence of polymer CF34 compared to the concentration after incubation for three days at 18°C in the presence of a control.
Figure 8c shows the concentration of malondialdehyde in spermatozoa after incubation for eight days at 18°C in the presence of polymer CF34 compared to the concentration after incubation for eight days at 18°C in the presence of a control.
Figure 9 shows the percentage of viable spermatozoa over an eight day period upon incubation with CF34 polymer, compared to a control.
Figure 10 shows the percentage of motile spermatozoa over an eight day period upon incubation with CF34 polymer, compared to a control.
Figure 1 : Within the 1H NMR spectrum (obtained in
CDC13) obtained at 200 MHz broad overlapping peaks dominated. Significant peaks are at delta 0.82-
0.85 (m, 3H3 sugar 3H9a, 3H5a, 3Hla, 3Hlb, 3H24 alpha-tocopherol), 3.80 (broad, 2H6, HEMA) , 4.99-
5.38 (broad, H8 , H9, H10, Hll, 2H12 galactose) identifying the polymer as containing alpha tocopheryl methacrylate, 2- (2 ' , 3 ' , 4 ' , 6' -tetra-O- acetyl-beta-D-galactosyloxyl) ethyl methacrylate and
2-hydroxyethyl methacrylate.
Figure 2 : Within the 1H NMR spectrum (obtained in D20) . Peaks of significance are at delta 0.71
(broad s, 3H9a, 3H5a, 3Hla, 3Hlb, alpha- tocopherol) , 3.04-3.08 (galactose ring protons) and 4.17 (broad, -CH2 -CH2 -OH, DMAEA) identifying the polymer as containing alpha tocopheryl acrylate, 2-
(beta-D-galactosyloxyl) ethyl acrylate and 2-
(dimethylamino) ethyl acrylate.
The compositions of all polymers prepared were determined by 1H NMR and are given in Table 1.
Table 1. Composition of terpolymers (wt . % of total) .
AcGalEA: 2- (2 ' , 3 ' , 4 ' , 6 ' -tetra-O-acetyl-beta-D- galactosyl) ethyl acrylate; GalEA: 2- (beta-D- galactosyl) ethyl acrylate; AcGalEMA: 2-
(2' ,3' ,4' , 6' -tetra-O-acetyl-beta-D-galactosyl) ethyl methacrylate; GalEMA: 2- (beta-D-galactosyl) ethyl methacrylate; alpha-TA: alpha-tocopheryl acrylate; alpha-TMA: alpha-tocopheryl methacrylate; HEMA: 2- hydroxyethyl methacrylate; DMAEA: 2-
(dimethylamino) ethyl acrylate; DMAEMA: 2-
(dimethylamino) ethyl methacrylate .
Cellular Application of the Polymeric Complexes. The efficacy of the complexes for the cellular delivery of lipophilic metabolites was tested using
a complex embracing various combinations of alpha- tocopherol, beta-D-galactose and DMAEA. The results described below are for two representative polymers (CF28 and CF34) with their proportions respectively of 20:20:60 (CF28) and 30:20:50 (CF34) by weight of alpha-tocopheryl acrylate, 2-(beta-D- galactosyl) ethyl acrylate and DMAEA. Spermatozoa cells of the pig were chosen for the evaluation of metabolite (alpha-tocopherol) delivery, its uptake and subsequent intracellular action, such cells being seen as presenting an extreme challenge for the measurement of alpha-tocopherol incorporation and action through their unique combination of a wholly aqueous environment, large medium (seminal fluid) volume per unit cell and high cellular concentration of highly oxidisable lipid substrate, long chain polyunsaturated fatty acids.
All ejaculates were obtained from standard commercial breeding boars housed according to UK/EU standards for artificial insemination centres. The boars were between 9 and 14 months of age and of high reproductive capacities. Diets fed were normal for breeding boars. Semen samples were obtained and processed "post ejaculation" and before in vitro experimentation according to standard commercial procedures.
Spermatozoa quality parameters e.g. concentration, motility, viability and chemical determinations (lipid/fatty acids, cellular metabolites, oxidation products, etc.) were quantified by established
analytical and commercial techniques as appropriate. Statistical analyses were applied where necessary and possible .
Table 2 shows the mean values for the major long chain polyunsaturated fatty acid levels within the spermatozoa used throughout the experimentation.
Table 2. Polyunsaturated fatty acid composition (major acids, wt . % of total fatty acids ±SE) of the spermatozoa. 1 = common name. 2 = short hand designation (chain length: number of double bonds, family) .
linolθic 1 (18:2 n-6) 2 2.1 ± 0.12 alpha-linolenic (18:3 n-3) 0.8 ± 0.02
Arachidonic (20:4 n-6) 3.0 ± 0.21
Docosapentaenoic (22:5 n-6) 24.8 ± 2.3
Docosahexaenoic (22:6 n-3) 33.1 ± 2.9 total polyunsaturates 63.8 ± 5.01
As can be seen the highly unsaturated long chain fatty acids docosapentaenoic (22:5n-6) and docosahexaenoic (22:6n-3) were both present at substantial levels and together accounted for some 60% of the total fatty acids present in the spermatozoa lipid, thereby presenting the high risk for lipid peroxidation and associated deleterious effects in standard commercial boar semen.
Of prime importance to the quantification of polymer uptake and potential functional effectiveness to deliver alpha-tocopherol is1 the ability to distinguish analytically within the spermatozoa cell between polymer bound and free alpha-tocopherol.
Figure 3 : The ability of the spermatozoa to differentiate between the two forms of alpha- tocopherol by means of high performance liquid chromatography is shown. Two major peaks eluting at 12.137 and 20.917 minutes after injection were identifiable as polymer bound and free alpha- tocopherol respectively by comparing their elution pattern with pure standards.
Figure 4 : The ability of a spermatozoa cell to take up the alpha-tocopherol polymer is compared to the ability of a spermatozoa cell to take up the alpha- tocopherol. Figure 4 also shows the amount of alpha-tocopherol released from its carrier once within the cell. It is evident from the data given in Figure 4 that alpha-tocopherol take up is increased through the use of the cellular delivery system of the present invention. Differing concentrations of polymer CF28 and spermatozoa cells were incubated in vitro for a single given time (2 hours) in a commercial pig semen diluent (BTS) .
Figure 5 : A dose dependency between the extent of alpha-tocopherol accumulation within the cell and
the level of polymer exposure is shown. The very high levels to which cellular alpha-tocopherol may be raised are apparent when spermatozoa exposure to the polymer is extended.
Figure 6 and 7 : The ability of the polymer to supply alpha-tocopherol to the spermatozoa is shown over a period of time commensurate with and beyond that for normal boar semen storage i.e. approximately 8 days. The spermatozoa is stored under appropriate commercial conditions. The achievement of a significant . difference in the level of uptake of alpha-tocopherol following exposure to polymer CF34 (30% by weight of alpha- tocopherol) compared to CF28 (20% by weight of alpha-tocopherol) is clear.
The value in practical terms to the spermatozoa of the ability of the polymers to deliver enhanced levels of alpha-tocopherol was quantified by reference to purely bench parameters (protection afforded against cellular oxidation and accumulation of oxidised metabolites under normal and induced pro-oxidation storage conditions) and practical in vivo parameters (spermatozoa concentration, viability and motility during commercial storage) .
Figure 8a: The evolution of malondialdehyde (a natural product and specific indicator of oxidation of tissue polyunsaturated fatty acids) is decreased significantly in commercially diluted semen after
incubation for 2 hours at 37°C with CF34 polymer. This was carried out under conditions of stimulated tissue oxidation by the addition of FeS04. The evolution of malondialdehyde to an even greater extent following a 3 -day (Figure 8b) or 8 -day (Figure 8c) incubation with CF34 polymer under commercial conditions at 18°C. As can be seen, the effect of the polymer under these extremes of storage was to reduce oxidation some 10-fold compared to control semen i.e. without polymer CF34.
Figures 9 and 10: Positive effects arising from the controlled delivery of alpha-tocopherol via polymer delivery on major practical parameters of spermatozoa reproductive capacities are shown.
Thus over an 8 -day storage period under conditions that accorded with standard commercial practice, significant beneficial effects were seen on three major aspects in particular which are routinely accepted as a measure of artificial insemination efficacy namely; the extent of damaged spermatozoa cells; overall spermatozoa motility; and their relative motility (inclusion of CF34 increased the relative motile rating throughout the 8 day storage period) .