Source: http://www.google.com/patents/US4866052?dq=Frischling
Timestamp: 2013-12-10 10:05:47
Document Index: 676797911

Matched Legal Cases: ['Application No. 83301660', 'Application No. 83', 'Application No. 478494', 'Application No. 8407180', 'Application No. 84301882', 'Application No. 84', 'Application No. 8427485', 'art 1', 'art 1']

Patent US4866052 - Treatment of sickle cell disease - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Advanced Patent Search | Sign inAdvanced Patent SearchPatentsNeutral 2:1 ligand:zinc(II) complexes in which at least one ligand is provided by a compound being 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms are of value for...http://www.google.com/patents/US4866052?utm_source=gb-gplus-sharePatent US4866052 - Treatment of sickle cell diseasePublication numberUS4866052 APublication typeGrantApplication numberUS 07/124,115PCT numberPCT/GB1987/000194Publication dateSep 12, 1989Filing dateMar 19, 1987Priority dateMar 20, 1986Fee statusLapsedAlso published asEP0263138A1, EP0263138B1, WO1987005509A1Publication number07124115, 124115, PCT/1987/194, PCT/GB/1987/000194, PCT/GB/1987/00194, PCT/GB/87/000194, PCT/GB/87/00194, PCT/GB1987/000194, PCT/GB1987/00194, PCT/GB1987000194, PCT/GB198700194, PCT/GB87/000194, PCT/GB87/00194, PCT/GB87000194, PCT/GB8700194, US 4866052 A, US 4866052A, US-A-4866052, US4866052 A, US4866052AInventorsRobert C. Hider, Ernst R. HuehnsOriginal AssigneeNational Research Development CorporationExport CitationBiBTeX, EndNote, RefManPatent Citations (4), Non-Patent Citations (20), Referenced by (22), Classifications (8), Legal Events (7) External Links: USPTO, USPTO Assignment, EspacenetTreatment of sickle cell diseaseUS 4866052 AAbstract Neutral 2:1 ligand:zinc(II) complexes in which at least one ligand is provided by a compound being 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms are of value for use in effecting an enhancement of the oxygen binding ability of a patient's haemoglobin, this being of particular application in the treatment of sickle cell disease.
We claim: 1. A method for enhancing the oxygen binding affinity of halmoglobin in a patient in need of such treatment, said method comprising treating the blood of said patient with an amount effective to achieve such enhancement of a neutral 2:I ligand:zinc(II) complex in which at least one ligand is provided by a compound which is 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms.
5. A method according to claim 3, in which the treatment of the blood is effected at a concentration of zinc in the form of the complex which is from 10.sup.-3 to 10.sup.-5 molar.
6. A method according to claim 5, in which the concentration is from 5
8. A method according to claim 1, in which each ligand of the complex is separately provided by a compound selected from the following compounds of types (1), (2) and (3):(1) 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms; (2) a 3-hydroxypyrid-2-one or 3-hydroxypyrid-4-one in which the hydrogen atom attached to the nitrogen atom is replaced by an aliphatic acyl group, by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, or by an aliphatic hydrocarbon group substituted by one or more substituents selected from aliphatic acyl, alkoxy, cycloalkoxy, aliphatic amide, aliphatic ester, halogen and hydroxy groups and, optionally, in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by one of said substituents, by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, or by an aliphatic hydrocarbon group substituted by an alkoxy, cycloalkoxy, aliphatic ester, halogen or hydroxy group; and (3) an alternative compound providing a physiologically acceptable monobasic, bidentate ligand which is capable of bonding covalently to zinc; but with the proviso that at least one ligand is of type (1). 9. A method according to claim 8, in which the compound of type (3) either contains a first grouping which is an enolic hydroxy group or a carboxy group and a second grouping which is an amine group or a hydroxy group, or is a monocarboxylic acid providing an anion containing a grouping ##STR7##
The 2:1 zinc complexes used in the present invention contain zinc in the divalent state and are neutral, there being an internal balance of charges between the metal cation and the two monobasic, bidentate ligands bound covalently thereto. The hydroxypyrones described above will provide such a monobasic, bidentate ligand by the loss of a proton from the hydroxy group (OH→0.sup.-). Although the complexes used in the present invention are required to contain at least one hydroxypyrone ligand, the second ligand may if desired be derived from any alternative compound which will provide a physiologically acceptable, monobasic, bidentate ligand which is capable of binding to zinc. The inclusion in a complex of two different ligands can produce an added dimension to the design of complexes having optimised properties for uptake by erythrocytes. S However, both ligands are more conveniently derived from a hydroxypyrone and, in general, complexes containing two identical hydroxypyrone ligands are preferred by virtue of their greater simplicity of preparation and use.
It will be appreciated that the zinc complexes used in the present invention may exist in either a tetrahedral or an octahedral form since, although complexes containing a 2:1 proportion of monobasic, bidentate ligand:zinc(II) will usually have the tetrahedral form, it is possible for them to adopt the octahedral form by combination with two additional neutral ligands, in particular water molecules. The neutral 2:1 complexes may conveniently therefore be used in either the anhydrous or the dihydrate form, the anhydrous form possibly being converted to the dihydrate form in an in vivo aqueous environment. (-&gt;
Examples of hydroxypyrones providing ligands which may be used in complexes according to the present invention have the formula (I), specific hydroxypyrones of particular interest having the formulae (II) and (III): ##STR2## in which R is an alkyl or cycloalkyl group, for example methyl, ethyl, n-propyl, isopropyl or butyl, and n is 0, 1, 2 or 3 (the ring being unsubstituted by any alkyl group when n is 0). Among these compounds 3-hydroxy-2-methyl-4-pyrone (maltol; II, R =CH.sub.3), 3-hydroxy-2-propyl-4-pyrone (II, R=CH.sub.2 CH.sub.2 CH.sub.3), 3-hydroxy-2-(2'-methylethyl)-4-pyrone (II, R =CH(CH.sub.3).sub.2) and especially 2-ethyl-3-hydroxy-4-pyrone (II, R=C.sub.2 H.sub.5) are of most interest, although 3-hydroxy-4-pyrone (I, n =0) and 3-hydroxy-6-methyl-4-pyrone (III, R=CH.sub.3) are also of especial interest.
The 3-hydroxypyrid-2-ones are generally of somewhat greater interest than the 3-hydroxypyrid-4-ones, due to the larger partition coefficients of zinc complexes containing ligands provided by compounds of the former group. Other detailed preferences relating to the nature and position of the substituent groups present in the hydroxypyridones.-. are broadly as expressed in relation to hydroxypyridone iron complexes in UK Patent 2117766 and in equivalent Applications (European Patent Application No. 83301660.3, Japanese Patent Application No. 83/049677, US Patent Application No. 478494, etc.) and in UK Patent Application No. 8407180, published as GB 2136806A, and in equivalent Applications (European Patent Application No. 84301882, Japanese Patent Application No. 84/057186, U.S. Pat. No. 592543, now U.S. Pat. No. 4,650,793, etc.). Thus substituted aliphatic hydrocarbon groups present in the hydroxypyridones may as indicated therein carry more than one substituent group, but it is preferred that only one substituent group is present. Such substituted aliphatic hydrocarbon group substituents may conveniently derive from aliphatic hydrocarbon groups of 1 to 8 and particularly of 1 to 6 carbon atoms. However, the simpler hydroxypyridones of UK Patent Application GB 2118176A containing only unsubstituted aliphatic hydrocarbon group substituents of 1 to 6 carbon atoms are of the greatest interest. The preferences among the aliphatic hydrocarbon groups present in these hydroxypyridones correspond largely to those expressed in relation to the hydroxypyrones, with methyl groups conveniently being used for substitution on ring carbon atoms but larger alkyl or cycloalkyl groups also being of Particular interest for substitution on the ring nitrogen atoms. Substitution of the ring carbon atoms, which is again preferably by one rather than two or three aliphatic hydrocarbon groups, is of particular interest in the case of the 3-hydroxypyrid-4-ones, for example at the 6- or particularly the 2-position, whilst the 3-hydroxypyrid-2-ones may more often be used without any additional aliphatic hydrocarbon group substituent on the ring carbon atoms. Specific hydroxypyridones of particular interest have formulae (VI), (VII), (VII), and (VIII) ##STR4## in which R is an alkyl group, for example methyl, ethyl, n-propyl, isopropyl or butyl, and R.sup.1 is hydrogen or particularly an alkyl group, for example methyl. Among such compounds 1-ethyl-3-hydroxy pyrid-2-one, 3-hydroxy-1-propylpyrid-2-one, 3-hydroxy-1-(2metyyl-ethyl)-pyrid-2-one, 1-butyl-3-hydroxypyrid-2-one, 1-ethyl-2-methyl-3-hydroxypyrid-4-one, 2-methyl-1-propyl-3-hydroxypyrid-4-one, 3-hydroxy-2-methyl-1-(2'-methylethyl)-pyrid-4-one and 1-butyl-hydroxy-2-methylpyrid-4-one are of particular interest with the 3-hydroxypyrid-2-ones such as 1-ethyl-3-hydroxypyrid-2-one being especially preferred.
Among the quite wide range of ligands described above certain ligands or combinations of ligands will be of particular value and some indication of these has already been given. One measure of the value of the different complexes is provided by the value of their partition coefficient (K.sub.part) between n-octanol and Tris hydrochloride (20 mM, pH 7.4; Tris representing 2-amino-2 hydroxymethylpropane 1,3-diol) at 20 this being expressed as the ratio (concentration in organic phase)/(concentration in aqueous phase). Preferred complexes show a value of K.sub.part for each ligand-providing compound of above 0.02 or 0.05 but less than 3.0, especially of above 0.2 but less than 1.0, together with a value of K.sub.part for the 2:1 zinc(II) complex of above 0.02 but less than 6.0, especially of 0.05 or 0.1 to 1
Reaction to form the zinc complex is generally rapid and will usually have proceeded substantially to completion after 5 minutes at about 20 C., although a longer reaction time may be used if necessary. Following separation of any precipitated by-product, such as a sodium chloride in the case of certain solvent systems, the reaction mixture may conveniently be evaporated on a rotary evaporator or freeze dried to yield the zinc complex which is usually in solid form. A solid complex may, if desired, be crystallised from a suitable solvent, for example water, an alcohol such as ethanol, or a solvent mixture, including mixtures containing an ether. Whether the zinc complex is obtained in anhydrous or dihydrate form will depend both on the solvent system used for the reaction and on the subsequent working up procedure. Thus rigorous drying may remove the water molecules from a hydrated complex formed in a reaction mixture containing water and recrystallisation from an aqueous medium may add them. If a dihydrate is specifically required, freeze drying of the mixture resulting from the reaction of the ligand-providing compound(s) and zinc salt such as the chloride or acetate in an aqueous/organic solvent medium may be employed.
As an alternative to the above procedures directed to the preparation of complexes in the solid state, aqueous solutions of zinc hydroxypyrone complexes, for example, may conveniently also be prepared directly by dissolving a zinc salt, for example zinc sulphate, and the hydroxypyrone in water and adjusting the pH of the solution to between 7.0 and 8.0, for example 7 4. Thus, the (addition of zinc sulphate (25 mmoles) to a solution of 2-ethyl-3-hydroxy-4-pyrone (25 (100 ml) leads to the complete chelation of the zinc when the pH is adjusted to 7.4 by the addition of a base, for example sodium hydroxide.
Although it has been found that an increase in concentration of the zinc complex used in the treatment of the patient's blood leads to an increase in the level of binding of zinc to the haemoglobin and consequently in the enhancement of the oxygen binding ability thereof, it has also been found that the use of the complex at too high a concentration level can have an effect on the erythrocytes which lowers their half life. This leads to the surprising result that the optimum concentrations of the zinc complex are quite low, the increased ability of the zinc complexes to produce a left shift of the oxygen dissociation curve, as described hereinafter, in the case of sickle erythrocytes as compared with normal erythrocytes being one factor which makes possible the use of such low concentrations. The preferred concentrations of the complex are most conveniently expressed in terms of the molar concentration of zinc (as the zinc complex) in the blood treatment medium in order to take account of the varying proportion by weight of zinc in complexes containing ligands of varying molecular weight, although even so the preferred range may depend somewhat upon the particular complex being used. As a guide, however, it may be stated that, although levels of as high as about 10.sup.-2 molar (10 mM) may be used with marked beneficial effect on the oxygen binding ability of the patient's haemoglobin, these levels will shorten the half life of the erythrocytes. Preferably, therefore, lower levels are used which have little or no such effect whilst retaining a measure of enhancement of oxygen binding ability, for example a concentration of zinc (as the zinc complex) of 10.sup.-3 to 10.sup.-5 molar. However, some slight reduction of half life has still been observed at a zinc concentration of 10.sup.-3 molar and a value less than this is therefore preferred, a convenient range for the concentration of zinc (as the zinc complex) being 5
desired, the upper limit can be reduced below 5 -; - 14 example to less than 2.5 or 2 similar lower limit giving a range, for example, of 10.sup.-4 molar to 10.sup.-5 molar. However, the effect on half life observed at 5 5
In terms of the amount of zinc in grams used to treat one litre of blood it will be appreciated that, as the controlling factor for optimisation of the treatment conditions is the concentration of zinc in the medium in which the red cells are treated, a conversion factor must take into consideration the difference in concentration of the red cells in the blood and in the medium. The reduction in volume from the former to the latter is usually of the order of one half so that, as a guide, it may be stated that a concentration of 10.sup.-4 molar usually corresponds approximately to the use of about 0.003 grams of zinc per litre of blood. Thus, although amounts of 0.2 to 0.4, for example 0.3, grams of zinc per litre of blood may be used, the desired aim of avoidance of a reduction of half life requires the use of lower amounts of about 0.03 to about 0.0003 grams of zinc per litre of blood, particularly about 0.015 to about 0.003 grams.
The zinc complex will usually be added to the cells in the form of a solution in a physiologically acceptable diluent, for example physiological saline Practical Immunology, Hudson and Hay (editors), Blackwell Scientific Publications, 1980, page 336 excess of the ligand forming compound or compounds in preventing dissociation of the 2:1 complexes under certain conditions has been fully discussed in UK Patent Application No. 8427485 and its equivalents. Although the extra-corporeal use of the complexes described herein, involving as it does essentially neutral conditions and relatively high concentrations, does not present as many problems from the point of view of dissociation of the complexes as does oral administration, it may nevertheless often be worthwhile employing the complex together with an excess of the free ligand-providing compound or compounds. A preferred range for the molar proportion of the free compound to zinc complex which is present when each ligand is identical is thus from 0 to 100 moles of free compound:1 mole of zinc complex. Conveniently, a proportion of up to no more than 50, 30 or especially 20 moles:1 mole is used with a lower level of 0.5, 1 or 2 moles:1 mole. A particularly preferred range is from 2 or 3 up to 18 or 20, or especially up to 30 or 50, moles of free compound: 1 mole of zinc complex, particularly 2 or 3, or especially 5, up to 8 or 10, especially up to 20, moles:1 mole. When there is more than one zinc bound ligand present in the composition, either in the form of a mixed ligand zinc complex and/or of a mixture of zinc complexes, then the proportion of each free ligand-providing compound to the zinc complex containing that ligand may conveniently also fall in the ranges indicated above. It will be appreciated, however, that when more than one ligandproviding compound is present it is less likely that the proportion of each will be towards the upper end of the broadest range of 0 to 100 moles of free compound:1 mole of zinc complex, the proportion being more likely to lie in a range of 0.5 to 50 moles:1 mole or such lower ranges as are quoted above. Indeed, the total molar concentration of both ligands (usually equimolar proportions) may often lie within the range indicated.
The use of the low concentrations of zinc complex which are suitable to avoid a reduction in the half life of the erythrocytes does preferably involve the use of an added amount of the free ligand or ligands to prevent undue dissociation of the complex at these low concentrations, the appropriate excess of free ligand increasing with a decrease in the concentration at which the zinc complex is used. Thus, in order to optimise zinc uptake by erythrocytes at a physiological pH of 7.4, preferred ranges for the ratio of moles of free compound or, where two different ligands are present, of both compounds in total (usually in equimolar proportions):1 mole of zinc (as the zinc complex) are as follows:- from 2:1 to 5:1 for 10.sup.-3 molar zinc; from 5:1 to 10:1 for 5 from 20:1 to 30:1 for 10.sup.-5 molar zinc.
BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1a, 1b, 2 and 3 graphically show results obtained using Zn complexes as claimed herein compared to a control.
EXAMPLE 1 Uptake of zinc from complexes by haemoglobin (1) A 5% suspension of human erythrocytes in aqueous sodium chloride (b 130 mM) buffered to pH 7.4 by 20 mM Tris hydrochloride (2 ml) was incubated for 1 hour at 37 with the 2:1 zinc(II) complex in the presence of an excess of the ligand-forming compound. The complex was formed in situ in the buffer using .sup.65 Zn labelled ZnSO.sub.4 at a concentration selected over a range from 1 this concentration and a small amount of Tris free base (to about 2 mM) added to the medium to avoid an acid pH on reaction of the ZnSO.sub.4 and the compound to form the complex. Following the period of incubation, an aliquot of the erythrocyte/medium mixture was placed above a layer of silicone oil and the erythrocytes separated by - 30 centrifugation through the oil. The .sup.65 Zn levels associated with the erythrocytes and the incubation medium were then counted and the distribution ratio (concentration in erythrocytes/concentration in medium) calculated. The average distribution ratios of zinc obtained in a series of experiments for the homogeneous 2:1 complex from the ligand-providing compounds 3-hydroxy-2methyl-4-pyrone, 2-ethyl-3-hydroxy-4-pyrone and 3-hydroxy-2-(1'-methylethyl)-4-pyrone at a concentration of 5 x 10-4M are shown in Table 1 together with the percentage of the total amount of zinc which is absorbed in the erythrocytes.               TABLE 1______________________________________Uptake of zinc by haemoglobin            Distribution                        Percentage ofLigand-providing compound            ratio       zinc absorbed______________________________________3-hydroxy-2-methyl-4-pyrone            8           542-ethyl-3-hydroxy-4-pyrone            300         963-hydroxy-2-(1'-methylethyl)-4-pyrone         550         98______________________________________
There was no evidence of any saturation of the rate of uptake over the concentration range studied so that the complex is not actively transported and, in order to account for the high distribution ratios observed, the zinc must therefore dissociate from the complex and bind to a macromolecule or membrane associated with the erythrocyte. The experiment just described was repeated at a concentration of 2.5 mM .sup.65 ZnSO.sub.4 and 25 mM 2-ethyl-3-hydroxy-4-pyrone using erythrocyte ghosts in which the haemoglobin is lacking rather than whole erythrocytes. Only a low level of uptake of zinc was observed indicating that membrane binding is not responsible for the high distribution ratios observed with the whole erythrocytes. (2) The ability of the zinc(II) complex of 2-ethyl-3-hydroxy-4-pyrone to donate zinc to haemoglobin was investigated by [j studying the elution profile of the .sup.65 Zn label when a mixture of haemoglobin and the .sup.65 Zn-labelled complex (at 1 mM concentration) in NaCI (130 mM) buffered to pH 7.4 by Tris hydrochloride is applied to a PD-10 colum (Sephadex G-10 gel permeation column - Pharmacia). It was found that, even when the free 2-ethyl-3-hydroxy-4-pyrone is present in an amount providing a 20-fold molar proportion of the ligand-providing compound:zinc, the zinc is found to bind tightly to haemoglobin.
EXAMPLE 2 Effect of the zinc complex of 2-ethyl-3-hydroxy-4-pyrone on the oxygen affinity of haemoglobin (1) A 50% suspension of human erythrocytes in pH 7.09 8BisTris/NaCl buffer005M BisTris(bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane and 0.13M NaCl was incubated for 15 minutes at 37 zinc(II) complex of 2-ethyl-3-hydroxy-4-pyrone in the presence of an excess of the ligand-forming compound, the complex being formed in situ using zinc sulphate and a 10 molar excess of 2-ethyl-3-hydroxy-4-pyrone (to provide the 2:1 complex and an 8 molar excess of the pyrone). The oxygen dissociation curve of the erythrocytes was then determined using the method of Bellingham and Huehns (Nature, -968, 218, 924-926) which involves measuring the change of OD at two wavelengths (570 and 555 nm) after the sequential measured addition of oxygen in a tanometer. The experiment was conducted using normal erythrocytes, 2,3-diphosphoglyceric acid (DPG) depleted erythrocytes, foetal erythocytes and sickle erythrocytes. A 2.5 mM concentration of zinc was employed in each case together with a proportion of erythrocytes such as to provide a 2:1 ratio of zinc:haemoglobin and consequent saturation of the haemoglobin by zinc, except in one experiment where the proportion was varied to provide a ratio varying from 0.5:1 to 2:1. In each case a control was carried out in the absence of any zinc sulphate or 2-ethyl-3-hydroxy-4-pyrone and in one case two additional controls were carried out in the presence of one of these compounds used alone. The values for p(O.sub.2)50 (i.e. the oxygen pressure at which 50% of the haemoglobin is in the oxygenated form) obtained in each case are shown in Table 2 (together with the inferred Zn:Hb ratio) from which it will be seen that the oxygen affinity of normal erythrocytes, and also of sickle erythrocytes, is increased through treatment of the cells with zinc(II) (2-ethyl-3-hydroxy-4-pyrone).sub.2. Foetal erythrocytes, which contain Hb-F and therefore show different characteristics from normal eythrocytes which contain Hb-A, were as expected not affected. The results show that with normal haemoglobin the zinc complex is producing a left-shift of the oxy-haemoglobin dissociation curve, i.e. the equilibrium in the reaction HbO.sub.2 "Hb+O.sub.2 has been displaced in favour of the HbO.sub.2 . With sickle erythrocytes the effect is even more marked as is further illustrated in (2) below.
(2) The effect of the 2:1 2-ethyl-3-hydroxy-4-pyrone:zinc(II) complex on normal and sickle erythrocytes was further compared using a basically similar procedure to that described in (-) above. Concentrations of 2.5 mM ZnSO.sub.4 and 25 mM pyrone, and a 2:1 Zn:Hb ratio were again used but the incubation period was extended to 1 hour. In each case a control experiment was carried out in the absence of any zinc sulphate or pyrone.
The results obtained are shown in FIG. 1, where (a) relates to the normal erythrocytes and (b) to the sickle erythrocytes, from which it will be seen that a significant left shift in the oxygen dissociation curve is produced in both cases but that the difference from the control is more marked for the sickle erythrocytes which are right shifted relative to the normal erythrocytes prior to treatment. Thus, the p(O.sub.2)50 values are control =27.5 mm Hg, treated cells =15.0 mm Hg (shift 12.5 mm Hg) for the normal
(3) The effect of different concentrations of the 2:1 2-ethyl-3-hydroxy-4-pyrone:zinc(II) complex upon normal erythrocytes was studied using a basically similar procedure to that described in (l) above but with ZnSO.sub.4 concentrations of 0.1, 1 and 10 mM (and ten times these concentrations of the pyrone) and extending the incubation period to 1 hour. A control experiment was carried out in the absence of any zinc sulphate or pyrone. The results obtained are shown in FIG. 2 from which it will be seen that although there is a dose response effect there is still a discernible left shift in the oxygen dissociation curve at a concentration of the complex of 0.1 mM.
EXAMPLE 3 Treatment of rabbit blood with zinc complex of 2-ethyl-3-hydroxy-4-pyrone Blood (4 ml) was venesected from the marginal ear vein of an adult New Zealand White rabbit and collected to obtain a final concentration of preservative free heparin of 20 international units/ml. The blood was washed with physiological saline and centrifuged at 2000 g to provide 0.5 ml of packed red blood corpuscles. These were incubated at 37 with an equal volume of either 2:1 2-ethyl-3-hydroxy-4-pyrone:zinc(II) together with an excess of the pyrone (0.1 mM ZnSO.sub.4 +2 mM pyrone) in physiological saline at pH 7.5 or of physiological saline alone at pH 7.5. After 30 minutes the cells were washed four times with physiological saline and then incubated at 37 labelled Na.sub.2 CrO.sub.4 in 0.5 ml physiological saline at pH 7.5-8 After 30 minutes incubation in this second medium the cells were washed three times with physiological saline and then resuspended in physiological saline to a final volume of 1 ml with 50% of final haematocite. This solution was then injected into the marginal ear vein of the rabbit from which 1 ml blood samples were venesected daily at 2 day intervals for determination of survival of the treated cells in vivo through measurement of the radioactivity of the .sup.51 Cr. erythrocytes and control =36.0 mm Hg, treated cells =18.5 mm Hg (shift 17.5 mm Hg) for the sickle erythrocytes.
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS4665064 *Oct 31, 1984May 12, 1987National Research Development CorporationPharmaceutical compositions and methods for increasing zinc levelsEP0022229A1 *Jun 27, 1980Jan 14, 1981The Wellcome Foundation LimitedSubstituted phenol ethers, their preparation, intermediates therefor, pharmaceutical compositions containing them and the preparation thereofEP0145228A1 *Oct 31, 1984Jun 19, 1985National Research Development CorporationPharmaceutical compositionsGB2148896A * Title not available* Cited by examinerNon-Patent CitationsReference1Arnone, A. et al, "The Binding of Zinc to Human . . .", Zinc Metabolism: Current Aspects in Health & Disease, 1977, pp. 317-328.2 *Arnone, A. et al, The Binding of Zinc to Human . . . , Zinc Metabolism: Current Aspects in Health & Disease, 1977, pp. 317 328.3Brewer G. J., "Interactions of Trace Elements . . .", Jour. of Ame. College of Nutrition 4:33-38 (1985).4 *Brewer G. J., Interactions of Trace Elements . . . , Jour. of Ame. College of Nutrition 4:33 38 (1985).5Brewer, G. J. et al, "Suppression of Irreversibly Erythrocytes by . . .", J. Lab. Clin. Med., vol. 90, No. 3, Sep. 1977, pp. 549-554.6Brewer, G. J. et al, "Suppression of Irreversibly Sickled . . .", J. lab. Chem. Med., Sep. 1977, pp. 549-554.7 *Brewer, G. J. et al, Suppression of Irreversibly Erythrocytes by . . . , J. Lab. Clin. Med., vol. 90, No. 3, Sep. 1977, pp. 549 554.8 *Brewer, G. J. et al, Suppression of Irreversibly Sickled . . . , J. lab. Chem. Med., Sep. 1977, pp. 549 554.9Brewer, G. J., "Detours of the Road to Successful . . .", Perspect Biol. Med., vol. 22, No. 2, Part 1, Winter 1979, pp. 250-272.10 *Brewer, G. J., Detours of the Road to Successful . . . , Perspect Biol. Med., vol. 22, No. 2, Part 1, Winter 1979, pp. 250 272.11Gilman, J. G. et al, "The Oxygen-Linked Zinc-Binding . . .", Biochem. J. (1978) 169, pp. 625-632.12 *Gilman, J. G. et al, The Oxygen Linked Zinc Binding . . . , Biochem. J. (1978) 169, pp. 625 632.13Oelshlegel, F. J., "Effect of Zinc on Increasing Oxygen . . .", Biochemical & Biophysical Res. Communications, vol. 53, No. 2, 17 Jul. 1973, Academic Press, Inc. pp. 560-566.14 *Oelshlegel, F. J., Effect of Zinc on Increasing Oxygen . . . , Biochemical & Biophysical Res. Communications, vol. 53, No. 2, 17 Jul. 1973, Academic Press, Inc. pp. 560 566.15Oelshlegel, Jr. G. J. et al, "Studies on the Interaction of . . .", Archives of Biochem. & Biophysics, 163, 742-748 (1974).16 *Oelshlegel, Jr. G. J. et al, Studies on the Interaction of . . . , Archives of Biochem. & Biophysics, 163, 742 748 (1974).17Schoomaker, E. B. et al, "Zinc in the Treatment of Homozygous . . . ", American Jour. of Hematology, vol. 1, No. 1, 1976, pp. 45-57.18 *Schoomaker, E. B. et al, Zinc in the Treatment of Homozygous . . . , American Jour. of Hematology, vol. 1, No. 1, 1976, pp. 45 57.19Weatherall, D. J. et al, "Haemolytic Anemia . . .", Oxford Textbook of Med., vol. 2, Sec. 13-Index 1983, pp. 19.62-19.66.20 *Weatherall, D. J. et al, Haemolytic Anemia . . . , Oxford Textbook of Med., vol. 2, Sec. 13 Index 1983, pp. 19.62 19.66.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS5108754 *Feb 8, 1991Apr 28, 1992Michael WilburnOrthomolecular method of treating sickle cell diseaseUS5177208 *Jan 13, 1992Jan 5, 1993Michael WilburnOrthomolecular method of treating sickle cell diseaseUS5626884 *Aug 18, 1995May 6, 1997Lockett; Curtis G.Treatment of sickle cell diseaseUS6251927 *Apr 20, 1999Jun 26, 2001Medinox, Inc.Methods for treatment of sickle cell anemiaUS6339080Dec 9, 1997Jan 15, 2002Vitra Pharmaceuticals Ltd.Iron compounds, compositions, methods of making the same and uses thereofUS6610702Aug 1, 2001Aug 26, 2003Gmp Oxycell, Inc.Ammonium salts of inositol hexaphosphate, and uses thereofUS6635631Oct 25, 2001Oct 21, 2003Vitra Pharmaceuticals, Ltd.Iron compounds, compositions, methods of making the same and uses thereofUS7087640 *Mar 14, 2003Aug 8, 2006Technology Commercialization CorpSubstance with sedative effectUS7135196Sep 10, 2001Nov 14, 2006Vitra Pharmaceuticals LimitedIron compositionsUS7312078Mar 18, 2005Dec 25, 2007Gamida Cell Ltd.Methods of controlling proliferation and differentiation of stem and progenitor cellsUS7344881Apr 18, 2003Mar 18, 2008Gamida Cell Ltd.Methods of expanding stem and progenitor cells and expanded cell populations obtained therebyUS7429489Oct 14, 2004Sep 30, 2008Gamida Cell Ltd.Methods of controlling proliferation and differentiation of stem and progenitor cellsUS7459569May 7, 2003Dec 2, 2008Vitra Pharmaceuticals LimitedMethod of forming iron hydroxypyrone compoundsUS7655225Feb 19, 2008Feb 2, 2010Gamida Cell, Ltd.Methods of expanding stem and progenitor cells and expanded cell populations obtained therebyUS7855075May 19, 2008Dec 21, 2010Gamida Cell Ltd.Methods of controlling proliferation and differentiation of stem and progenitor cellsUS7955852Feb 9, 2004Jun 7, 2011Gamida Cell Ltd.Expansion of renewable stem cell populationsUS8080417Nov 29, 2006Dec 20, 2011Gamida-Cell Ltd.Methods of ex vivo hematopoietic stem cell expansion by co-culture with mesenchymal cellsUS8202724Oct 6, 2010Jun 19, 2012Gamida Cell Ltd.Methods of controlling proliferation and differentiation of stem and progenitor cellsEP1256342A1 *Nov 13, 2000Nov 13, 2002Japan Science and Technology CorporationHypoglycemics comprising organic zinc (ii) complexesEP1634591A2 *Nov 13, 2000Mar 15, 2006Japan Science and Technology AgencyHypoglycemics comprising organic zinc (II) complexesWO1998050051A1 *May 5, 1997Nov 12, 1998Curtis LockettTreatment of sickle cell disease, treatment of immune system diseases and other diseases normally associated with sickle cell anemiaWO2000062777A1 *Apr 6, 2000Oct 26, 2000Chen Long ShiuhMethods for treatment of sickle cell anemia* Cited by examinerClassifications U.S. Classification514/184, 514/188International ClassificationA61K31/315, A61K31/555, A61P7/06, A61P7/00Cooperative ClassificationA61K31/315European ClassificationA61K31/315Legal EventsDateCodeEventDescriptionNov 25, 1997FPExpired due to failure to pay maintenance feeEffective date: 19970917Sep 14, 1997LAPSLapse for failure to pay maintenance feesApr 22, 1997REMIMaintenance fee reminder mailedFeb 25, 1993FPAYFee paymentYear of fee payment: 4Aug 11, 1992ASAssignmentOwner name: BRITISH TECHNOLOGY GROUP LIMITED, ENGLANDFree format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:NATIONAL RESEARCH DEVELOPMENT CORPORATION;REEL/FRAME:006243/0136Effective date: 19920709Jul 14, 1989ASAssignmentOwner name: NATIONAL RESEARCH DEVELOPMENT CORPORATION, ENGLANDFree format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:HIDER, ROBERT C.;HUEHNS, ERNST R.;REEL/FRAME:005123/0984Effective date: 19871026Jul 14, 1989AS02Assignment of assignor's interestOwner name: HIDER, ROBERT C.Owner name: HUEHNS, ERNST R.Effective date: 19871026Owner name: NATIONAL RESEARCH DEVELOPMENT CORPORATION, 101 NEWRotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google