Patent Abstract:
meso - zeaxanthin compositions for pharmaceutical use and use of meso - zeaxanthin to increase the deposition of macular pigment in the human eye , and for the therapeutic treatment or prophylaxis of diseases and disorders of the macula , in particular age - related macular degeneration .

Detailed Description:
the following examples are intended to illustrate the invention by way of example only , and are not intended to limit the scope of the invention . an hplc analysis of retinas obtained from normal and amd individuals was conducted using a sufficiently large sample to warrant conclusions on the importance of macular lutein and zeaxanthin in the prevention of amd . the amount and distribution of the macular carotenoids , including the stereoisomers . were determined and compared for 15 normal and 22 amd eyes in order to determine if there is evidence for or against the hypothesis that macular pigment protection from light exposure plays a significant role in reducing amd . for each normal and amd eye , the neural retina was cut into a central disk and 2 concentric annuli using trephines of 3 , 11 and 21 mm . to extract the carotenoids , the tissues were ground in ethanol / water ( 1 : 1 ) to which 10 ng lutein monomethyl ester was added as an internal standard . separation and quantitation of zeaxanthin and lutein fractions was by reversed - phase hplc using phenomenex column . carbamate derivatives of individual stereoisomers of both zeaxanthin and lutein were separated on a normal - phase hplc column , using the methods of ruttiman et al ( 1983 ) and schiedt et al . ( 1995 ), the results being plotted in fig2 . as shown in table 1 , amd eyes had on average approximately 70 % of the total carotenoid found in controls , a figure that was very consistent across the retina . seventeen ( 77 % of the twenty two amd eyes had total amounts of lutein and zeaxanthin in the central 3 mm of the retina which were below the mean 5 . 9 pmole / mm2 ) for the control group . for the two annuli , having outer diameters of 11 and 21 mm respectively , 15 ( 68 %) of the amd group were found to be lower in total carotenoids than the corresponding regions in the control group . the differences observed between the control and amd eyes in the inner annuli were found to be statistically significant ( on a one sided test p & lt ; 0 . 05 ); the difference in the medial and outer annuli were found almost significant ( p & lt ; 0 . 1 ). the relative distributions of carotenoids throughout the retina for normal and amd eyes were found to be essentially the same . both groups were characterized by a decrease in the quantity of meso - zeaxanthin and a relative increase in lutein with increasing distance from the fovea . the relative amounts of lutein and meso - zeaxanthin as compared to zeaxanthin are consistently lower in the amd retinas as compared to normal retinas . a trial was conducted to determine if dietary supplementation with lutein and zeaxanthin effectively can change the pigment levels in the macula . the optical density of the macula pigment was measured for each subject using the method of flicker photometry ( bone and sparrock 1971 ; bone et al 1992 ). the concentration of pigment in the macula is proportioned to its optical density and the actual amount of pigment was assumed to be proportional to concentration . thus , optical density was taken as a measure of the total amount of pigment . two healthy adult males ( of age / weight 42 year / 60 kg and 51 years / 61 kg ) ingested the equivalent of 30 mg of lutein per day in the form of lutein esters ( source : marigold flowers ) suspended in 2 ml of canola oil . this was continued over a period of 138 days and then the dose of lutein was discontinued . chemical analysis has shown that the product contains approximately 97 % lutein — and 3 % zeaxanthin . fasting serum lutein / zeaxanthin levels of both individuals were determined by conventional hplc on the morning of the first dose as a measure of base line . blood samples were drawn at 2 - 3 hour intervals throughout the first day for both subjects and then daily for the next three days . following the first week of supplementation , blood samples were drawn weekly . blood was collected into a standard vacutainer serum separator tube containing no anticoagulant . after allowing about 30 min for coagulation , the sample was centrifuged for 10 minutes and the serum removed by pipette . serum samples were stored at − 20 ° c . prior to analysis . carotenoids ware extracted from the serum by a minor modification of widely used methods ( guiliano et al , 1993 ; handelmann et al , 1992 ), 200 μl aliquots of serum were diluted with 2 ml of 50 % ethanol / water to ensure precipitation of protein components . 20 μl of an internal standard , monohexyl lutein ether , containing about 90 ng , was added to the solution at this point for quantification of the caratenoids by hplc . this solution was extracted 3 times with 2 ml portions of hexane by vortexing the sample for 1 min followed by centrifuging for 5 minutes and pipetting off the hexane layer . the three portions of hexane were dried under a stream of nitrogen gas and stared under nitrogen at − 20 ° c . until analysis was completed . serum extracts were dissolved in 4 . 0 μl of ethanol prior to injection . samples were vigorously agitated on a vortex mixer for 1 min to ensure dissolution of the sample . two replicate analyses were carried out using 20 μl aliquots . serum carotenoids were eluted at a flow rate of 1 ml / min through a 15 cm × 4 . 6 mm . adsorbosphere ods 3 μm hs column ( alltech ) coupled to a 25 cm × 4 . 6 mm spherisorb ods 5 μm column ( keystone scientific ) with detection of the carotenoids at 451 nm . [ 0058 ] fig3 a and 3 b show the increase in serum lutein concentration in the two subjects during the time course of the supplementation experiment . the concentration of lutein in both subjects increased by a factor of about 10 times within the first week and remained high thereafter . two other subjects llm and keg each receiving 30 mg / day lutein ester also showed large increases in serum lutein concentrations . subject llm increased from 0 . 5 μg / m to 1 . 20 μg / ml in 50 days and then reached a plateau . subject kes increased from 0 . 20 μg / ml to 1 . 22 μg / ml in 50 days . the optical density of the macular pigment was measured for each subject using the method of heterochomatic flicker photometry ( bone and sparrock , 1971 ; bone et al , 1992 ). the concentration of pigment in the macula is proportional to its optical density and the actual amount of pigment was assumed to be proportional to concentration . thus optical density was taken to be a measure of the total amount of pigment . in the flicker method , a small visual stimulus is presented to the eye which alternates in wavelength between 460 nm , the peak absorbance wavelength of the macular pigment , and 540 nm where pigment absorbance is zero ( bone et al ., 1992 ). above a certain frequency , color fusion occurs but the stimulus continues to flicker . at a higher frequency , a critical condition can be reached where flicker can be eliminated only if the two wavelength components are matched in luminance . if the stimulus is viewed peripherally , so that the image falls outside the macula , neither wavelength is attenuated by the macular pigment . however , if the stimulus is viewed centrally , the intensity of the 460 nm light must be increased to compensate for absorption by the macular pigment in order to achieve a luminance match . thus it is possible to determine the optical density of a subject &# 39 ; s macular pigment at the peak wavelength , or indeed any other wavelength . the validity of this technique depends on the relative spectral response of the receptors being the same in the central and peripheral locations used . the flicker , which the subject seeks to eliminate , is one of luminance and , assuming phototopic conditions , luminance is most likely due to an additive response from the long and middle wavelength sensitive cones ( guth et al , 1980 ). there is evidence that these two cone types are present in equal ratios in the two locations used ( wooten and wald , 1973 ). the short wavelength cones , whose relative abundances differ between the two locations , are generally not assumed to contribute to luminance ( guth et al , 1980 ), though others , using flicker techniques , have sought to eliminate their participation ( pease at al , 1987 ; werner et al . 1987 ; hammond et al 1995 ). the ultimate justification for our procedure is to be found in the accurate reproduction of the macular pigment absorbance spectrum which it generates ( bone et al , 1992 ). the apparatus consisted of a two - channel maxwellian view system based on a single light source , a 75 w xenon arc lamp . the wavelengths of the two channels were determined by 460 nm and 540 nm interference filters respectively , having half - widths of 7 and 9 nm . the channels were combined by a rotating semicircular mirror , and a circular aperture in a white screen provided a 1 . 5 ° diameter stimulus . cross - hairs facilitated central fixation of the stimulus . the screen was 18 ° in diameter and was illuminated with white light from the same source . the illuminance of the screen was adjusted to provide the same retinal illuminance of 4 log td as the stimulus . this was considered to be sufficiently high to minimize proteins associated with rod intrusion which could otherwise differentially affect measurements in the macula and peripheral retina ( wyszcki and stiles , 1982 ). a small red led was located 8 ° above the centre of the stimulus to provide a fixation mark for peripheral viewing of the stimulus . the intensity of the 460 nm channel was adjustable by the subject through a neutral density , compensated wedge whose setting could be recorded by a push - button . the flicker frequency was also under the subject &# 39 ; s control via a potentiometer . an adjustable dental impression bite ensured accurate and steady positioning of the subject &# 39 ; s eye relative to the exit pupil . the flicker frequency was set to a pre - determined value which , for central viewing by the subject , would allow flicker to be eliminated only over a very small range of wedge settings . this frequency was in the 25 to 35 hz range . having set the wedge to meet the no - flicker condition , the subject adapted to the viewing conditions by fixating with one eye on the stimulus cross - hairs for two minutes . the subject &# 39 ; s other eye was occluded by an eye - patch . at the end of this period , the subject proceeded to make a series of 10 to 15 wedge settings , attempting to obtain the center of the no - flicker range . the wedge was randomly offset after each setting . on occasions , the subject could not eliminate flicker entirely but instead sought a condition of minimum flicker . this was followed by another series of 10 to 15 settings while fixating on the led , the frequency having been reduced to 12 to 16 hz in order to reduce the range of no - flicker . the whole procedure was then repeated for the subject &# 39 ; s other eye . the optical density of the macular pigment of the subject was measured daily for a period of one week prior to the commencement of lutein supplementation and daily thereafter . [ 0067 ] fig4 a and 4 b show the absorbancy of the macular pigment during the time course of the experiments in the two subjects . an increase in the macular pigment level of subject jtl was first observable on the 14th day of supplementation . this subject had macular pigment levels in both eyes that were experimentally determined by repeated measurements to be equal (± 2 %) the initial values of 0 . 57 and 0 . 58 being determined by averaging 15 measurements obtained over a 17 day time period . comparison of these values with the average of 15 measurements obtained over the 18 day period at the end of the experiment gave values of 0 . 67 and 0 . 70 for the right and left eye , respectively , showing that the increase in optical density of the macular pigment is highly significant p & lt ; 0 . 0005 for both the right and left eye , based on a one sided t test . after discontinuing the dose of lutein at day 138 , optical density continued to rise until about day 180 and then reached a plateau . for subject rab , right and left eyes were found to have significantly different macular pigment density . the initial mean value for the right eye was 0 . 66 while that of the left eye was 0 . 76 . this corresponded to a difference of 15 % between the subject &# 39 ; s left and right eyes . the increase in macular pigment determined by comparing the initial averages for each eye and the final average ( 0 . 70 right , 0 . 79 left ) was found to be highly significant ( p & lt ; 0 . 001 ). after discontinuing the dose at day 138 , the optical density continued to rise in both eyes until day 200 and then reached a plateau . after several weeks of administration of lutein , the palms of the hands of each subject turned a noticeable yellow colour . this condition is similar to that induced by beta - carotene at the same dose . two other subjects llm and kes receiving 30 mg / day lutein ester were examined . for subject llm after 75 days the macular pigment had increased in both eyes from 0 . 3 to 0 . 5 optical density and then reached a plateau . for subject kes , the data had a much larger spread of values and an increase was only apparent in the left eye of optical density from 0 . 3 to 0 . 5 . macular pigmentation increase was shown to be a slow process , despite the high plasma lutein levels . this may be partly due to the need for lutein to diffuse into the avascular macular region of the retina . one would expect meso - zeaxanthin to be as active or more active than lutein . the trial established a relationship between the increased serum levels of lutein and corresponding increases in the concentration of lutein in the macula of the human eye . long term lutein supplement of individuals having low levels of macular pigmentation could result in a significant increase in the level of pigmentation within the macula . our data suggests that macular pigmentation does function to protect the retina . an increased rate of photo oxidation might accompany lower macular pigment levels in some individuals and could contribute to a more rapid build up of pathological lesions associated with amd . a capsule was prepared using the following ingredients by simple admixture and routine encapsulations : ingredients per capsule mg per capsule meso - zeaxanthin 10 lecithin 50 soya bean oil 200 a capsule was prepared using the following ingredients by simple admixture and routine encapsulation : ingredients per capsule mg per capsule lutein ester 75 ( equivalent to 10 mg lutein ) meso - zeaxanthin 75 lecithin 25 soya bean oil 100 a capsule was prepared using the following ingredients by simple admixture and routine encapsulation : ingredients per capsule mg per capsule vitamin c ( ascorbic acid ) 160 α - tocopheral 110 meso - zeaxanthin 90 lecithin 25 soya bean oil 75 a suitable daily dose for treatment amd would be one - two capsules daily . the procedure of example 7 was repeated except that 30 mg of coenzyme q10 was included in the mixture . a size 12 oval capsule of nominally 800 mg weight was prepared from the following ingredients by simple admixture and routine encapsulation : a dry powder formula diet composition was prepared by mixing 5 - 10 mg of meso - zeaxanthin with a cambridge diet ( the cambridge diet is a registered trade mark ) product obtained from cambridge health plan ltd ., norwich , england under the product identification . retinas were first cut be circular punches into an inner disc ( inner annulus ) of 3 mm diameter which encompassed most of the macular retina , a medial reing ( medial annulus ) encompassing the retinal tissues between 3 and 11 mm in diameter , and an outer ring ( outer annulus ) encompassing the retinal tissues between 11 and 22 mm in diameter . table 3 contains data derived from these retinal analyses . from these data it can be concluded that individuals suffering from this disease had much less lutein and zeaxanthin in their retinas than individuals not suffering from this disease . in most cases , the medial and outer annuli lie outside the macular area , and , as such , would not generally be affected by this disease . for amd eyes , the reduced concentrations of lutein and zeaxanthin in areas outside the macula ( medial and outer annuli ) are clear evidence that these eyes suffered from a deficiency in these compounds and that the reductions measured were not simply a sequellae of the disease itself . the concentrations of both lutein and total zeaxanthin are reduced in the eyes of individals reportedly suffering from age - related macular degeneration . when the total zeaxanthin in the retina was further analyzed , the data demonstrated that retinal zeaxanthin is a mixture of the three isomeric forms ( 3r , 3 ′ r - zeaxanthin , 3r , 3 ′ s -( meso )- zeaxanthin and 3s , 3 ′ s - zeaxanthin ). the concentration of 3s , 3 ′ s - zeaxanthin is barely detectable in any annulus , and is , therefore , negligible for the purposes of this declaration . however , the concentration of meso - zeaxanthin is substantial . table 4 summarizes the results of comparative analyses between non - armd ( control ) retinas and retinas afflicted with age - related macular degeneration . as can be seen from the data , meso - zeaxanthin is reduced by 30 % in the inner annulus of amd eyes , by 14 % in the medial annulus and by 17 % in the outer annulus . fig6 - 8 show this data in graphic format for the inner , medial and outer annuli , respectively . the data in table 4 and in fig8 demonstrate that the concentration of r , r - zeaxanthin in the outer annulus is actually greater in amd eyes than in control eyes . this is both counter - intuitive and totally unexpected in light of other data for the same set of eyes ( e . g . concentrations of lutein and total zeaxanthin ). in addition , the mean concentrations of meso - zeaxanthin in these annuli are 17 % lower in amd eyes than in the controls . in fact , the loss of meso - zeaxanthin in amd eyes is greater than the loss of r , r ′- zeaxanthin across all annuli . it is evident from these data that meso - zeaxanthin contributes significantly to the differences measured in total zeaxanthin given in table 3 , while the natural form of zeaxanthin ( r , r ′) does not . the surprising and unexpected selective reduction in meso - zeaxanthin in the macula of amd eyes would not have been predicted from dietary and blood studies . the results suggest that treatment of amd by administration of meso - zeaxanthin should require much lower dosages than would be necessary for the other carotenoid compounds such as lutein . it is expected that amounts as low as 0 . 5 mg per day could be effective in the prevention or treatment of amd . dosages between 0 . 5 and 50 mg / day , preferably 1 - 20 mg / day , most preferably 5 - 15 mg / day are expected to be effective . publications cited herein are hereby incorporated by reference and are listed below for convenience . bone , r . a . and landrum , j . t . 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