United States Patent t19
`Allen et al.
`
`US005563126A
`till Patent Number:
`[45] Date of Patent:
`
`5,563,126
`* Oct. 8, 1996
`
`METHOD FOR TREATMENT AND
`PREVENTION OF DEFICI:ENC[ES OF
`VITAMINS B12, FOLIC ACID, AND B6
`
`Primary Examiner Raymond Henley, III
`
`Attorney, Agent, or Firm~Davis, Graham & Stubbs, L.L.C.
`
`Inventors: Robert H. Allen, Englewood; Sally P.
`Stabler, Denver, both of Colo.
`
`[57]
`
`ABSTRACT
`
`[54]
`
`[75]
`
`[73]
`
`Assignee: Metabolite Laboratories, Denver,
`Colo.
`
`Notice:
`
`The portion of the term of this patent
`subsequent to Dec. 20, 2011, has been
`disclaimed.
`
`[21] Appl. No.: 999,499
`
`[22] Filed:
`
`Dec. 29, 1992
`
`Related U.S. Application Data
`
`[63]
`
`Continuation-in-part of Set. No. 727,628, Jul. 10, 1991, Pat.
`No. 5,374,560, which is a continuation-in-part of Set. No.
`333,124, Apr. 3, 1989, abandoned, and Set. No. 345,885,
`May 1, 1989, abandoned, which is a continuation-in-part of
`Ser. No. 933,553, Nov. 20, 1986, Pat. No. 4,940,658.
`
`[51]
`
`Int. CI.~ ......................... A61K 31/70; A61K 311495;
`A61K 31/44
`[52] U.S. C1 .............................. 514152; 514/249; 514/345
`[58] Field of Search .............................. 514/52, 249, 345,
`514/814
`
`[56]
`
`References Cited
`
`A method tbr orally administering vitamin preparations is
`
`described which combine vitamin B12 (B lz, cobalamin) and
`
`folic acid (folate), with and without pyridoxine (B6), for
`
`preventing and treating elevated serum homocysteine (HC),
`
`cystathionine (CT), methylmalorfic acid (MMA), or 2-me-
`
`thylcitric acid (2-MCA) levels. These metabolites have been
`
`shown to be indicative of Ba2 and/or folic acid deficiencies.
`
`Further, it is likely that a B6 deficiency may be present with
`
`a B12 or folate deficiency. ]]ae method of the invention is
`
`also for use in lowering serum HC, CT, MMA, or 2-MCA in
`
`patients with or at risk for neuropsychiatric, vascular, renal
`
`or hematologic diseases. One embodiment of the invention
`
`is the use of a non-prescription formulation containing 2.0
`
`mg B~z and 0.4 mg folic acid, with and without 25 mg B~.
`
`Another embodiment uses a prescription strength formula-
`
`tion containing 2.0 mg Ba2 and 1.0 mg folic acid, with and
`
`without 25 mg B6. The method of the present invention
`
`eliminate the cosily and time consuming steps of distin-
`
`guishing between vitamin deficiencies once a deficiency is
`
`found by measurement of serum metaholite levels. The
`
`U.S. PATENT DOCUMENTS
`
`present invention is of particular benefit to the populations
`
`4,940,658
`4,945,083
`5,374,560
`
`7/1990 Allen et al .................................. 435/4
`7/1990 Jansen, Jr. ................................. 514/52
`12/1994 Allen et al .............................. 436/129
`
`at risk for elevated serum metabolite levels, such as the
`people over the age of 65, and populations that have or are
`
`at risk for neuropsychiatric, vascular, renal and hematologic
`
`OTHER PUBLICATIONS
`
`diseases.
`
`Gilman et al. ’q’he Pharmacological Basis of Therapeutics",
`Published 1980 By MacMillan (pp. 1333-1340).
`Barness, American Journ. of Clin. Nutr. (20) 1967 pp.
`573-577.
`
`10 Claims, 11 Drawing Sheets
`
`Sandoz Inc.
`Exhibit 1018-0001
`
`JOINT 1018-0001
`
`

`
`U.S. Patent
`
`Oct. 8, 1996
`
`Sheet I of 11
`
`5,563,126
`
`Elderly Outpatients, Age 65-99 (H-152)
`
`Control Subjects, Age 17-65 (N-IO0)
`
`3O
`
`10
`
`0
`100 2OO
`I
`I
`99 199 299
`
`300 400 500 600 700 800
`I
`I
`I
`I
`I
`I
`399 499 599 699 799 899
`
`900
`~ ~1000
`999
`
`Serum Cobalamin (pg/ml)
`
`Sandoz Inc.
`Exhibit 1018-0002
`
`JOINT 1018-0002
`
`

`
`U.S. Patent
`
`Oct. 8, 1996
`
`Sheet 2 of 11
`
`5,563,126
`
`10,000
`
`5,000
`
`2,000
`
`1,000
`
`500
`
`200
`
`100
`
`5O
`
`2O
`
`oo
`o¯
`¯
`
`+3SD
`
`+2SD
`
`-~--~el-- -- +3SD
`
`~o ==
`
`+2SD
`
`O0
`
`O0
`
`°o¯
`¯
`¯
`
`-2SD
`
`-2SD
`
`o_.
`o
`
`~
`
`o
`
`o
`
`oo
`°°
`0
`
`Control Subjects
`(N=10/100)
`
`Elderly Outpatients
`(N=38/152)
`
`FIG. 2
`
`Sandoz Inc.
`Exhibit 1018-0003
`
`JOINT 1018-0003
`
`

`
`U.S. Patent
`
`oct. 8, 1996
`
`Sheet 3 of 11
`
`5,563,126
`
`8O
`
`7O
`
`~, 4o
`
`10
`
`0
`
`000
`0000
`
`+3SD
`
`+2SD
`
`-2SD
`
`O0
`.... ¯ -----+3SD
`
`+2SD
`
`-2SD
`
`¯
`
`Control Subjects
`(N=10/100)
`
`Elderly Outpatients
`(N=38/152)
`
`FIG, ,_%
`
`Sandoz Inc.
`Exhibit 1018-0004
`
`JOINT 1018-0004
`
`

`
`U.S. Patent
`
`Oct. 8, 1996
`
`Sheet 4 of 11
`
`5,563,126
`
`6000-
`
`2000
`
`200
`
`100
`
`5O
`
`Pre Cbl
`
`Post Cbl
`
`FIG.
`
`Sandoz Inc.
`Exhibit 1018-0005
`
`JOINT 1018-0005
`
`

`
`U.S. Patent
`
`oct. 8, 1996
`
`Sheet 5 of 11
`
`5,563,126
`
`Homocysteine (umol/L)
`
`~ 0 0 0 0 0 0
`I I I I
`
`I
`I
`I
`
`FIG. 5
`
`Sandoz Inc.
`Exhibit 1018-0006
`
`JOINT 1018-0006
`
`

`
`U.S. Patent
`
`oct. 8, 1996
`
`Sheet 6 of 11
`
`5,563,126
`
`I Nursing Home Patients (N=-212)
`
`[] Co~Wol Subjects (N=-100)
`
`30
`
`2O
`
`~ 10
`
`0
`
`0
`
`99
`
`100 200 300 400 500 600 700 800 900
`
`199 299 399 499 599 699 799 899 999
`Serum Cobalarrm (l:)g/ml)
`
`~1000
`
`FIG. 6
`
`Sandoz Inc.
`Exhibit 1018-0007
`
`JOINT 1018-0007
`
`

`
`U.S. Patent
`
`Oct. 8, 1996
`
`Sheet 7 of 11
`
`5,563,126
`
`so,ooo-
`
`20,000
`
`10,000
`
`5,000
`
`2,000
`
`1,000
`
`500
`
`200
`
`100
`
`5O
`
`2O
`
`o
`
`+35D
`+2SD
`
`.~3SD
`+2SD
`
`oo
`o
`oo
`oO
`0
`
`-2SD
`
`-2SD
`
`Control
`Subjects
`(N= 101100)
`
`Nursing Home
`Patients
`(N=29/212)
`
`FIG. 7
`
`Sandoz Inc.
`Exhibit 1018-0008
`
`JOINT 1018-0008
`
`

`
`U.S. Patent
`
`oct. 8, 1996
`
`Sheet 8 of 11
`
`5,563,126
`
`100
`
`90
`
`8O
`
`-’- 7O
`
`6O
`
`5O
`
`4O
`
`E
`
`3O
`
`20
`
`10
`
`+3SD
`
`+2SD
`
`=¯
`
`+2SD
`
`Oo0
`O0oO
`
`o
`
`¯000~¯0:000
`¯
`
`-2SD
`
`_=
`
`-2SD
`
`Control
`Subjects
`(N=t0/100)
`
`Nursing Home
`Patients
`(N=29/212)
`
`Sandoz Inc.
`Exhibit 1018-0009
`
`JOINT 1018-0009
`
`

`
`U.S. Patent
`
`Oct. 8, 1996
`
`Sheet 9 of 11
`
`5,563,126
`
`20,000-
`
`10,000
`
`5.000
`
`~., 2,000
`
`1,000
`
`50O
`
`2OO
`
`100
`
`2O
`
`+3SD
`+2SD
`
`-2SD
`
`Pre Cobalamin Post Cobalamin
`
`FIG.
`
`Sandoz Inc.
`Exhibit 1018-0010
`
`JOINT 1018-0010
`
`

`
`U.S. Patent
`
`Oct. 8, 1996
`
`Sheet 10 of 11
`
`5,563,126
`
`100
`
`7O
`
`5O
`
`4O
`
`3O
`
`2O
`
`10
`
`+3SD
`
`+2SD
`
`-2SD
`
`Pre Cobalamin Post Cobalarnin
`
`FIG. I0
`
`Sandoz Inc.
`Exhibit 1018-0011
`
`JOINT 1018-0011
`
`

`
`U.S. Patent
`
`Oct. 8, 1996
`
`Sheet 11 of 11
`
`5,563,126
`
`I Framingt~am Sul3jects, Age 65-99 (N=_548)
`
`~-] Cor~ot Suiojects, Age 22-63 (N=117)
`
`3O
`
`2O
`
`10
`
`0
`
`99
`
`100
`
`199
`
`200 300 400 500 600 700 800 900
`
`299 399 499 599 699 799 899 999
`
`~10o0
`
`Serum Cob~larrm (pg/~)
`
`Sandoz Inc.
`Exhibit 1018-0012
`
`JOINT 1018-0012
`
`

`
`5,563,126
`
`1
`METHOD FOR TREATMENT AND
`PREVENTION OF DEFICIE, NCIES OF
`VITAMINS B~2, FOLIC ACID, AND B6
`
`The present application is a continuation-in-part of appli- 5
`cation no. 07/727,628 filed Jul. 10, 1991, now U.S. Pat. No.
`5,374,560 which is a continuation-in-part of application no.
`333,124 filed Apr. 3, 1989, now abandoned, and application
`no. 345,885 filed May 1, 1989, now abandoned, which is a
`continuation-in-part of application no. 933,553 filed Nov. 10
`20, 1986 and now issued as U.S. Pat. No. 4,940,658 on Jul.
`10, 1990.
`
`FIELD OF THE INVENTION
`
`This invention relates to the field of nutrition. Specifically,
`the invention is comprised of new oral vitamin preparations
`combining vitamin B12 (BI~, cobalamin) and folic acid
`(folate), and vitamin B12, folate, and pyddoxine (B~) for use
`in patients with elevated serum metabolite levels of
`homocysteine (HC), cystathionlne (CT), methylmalonic
`acid (MMA), or 2-methylcitric acid (2-MCA). The elevation
`of these metabolites has been shown to be indicative of
`tissue deficiencies of B 12 and/or folate and/or B6, and related
`to increased risk of neuropsychiatric, vascular, renal and
`hematologic diseases. One embodiment of the present
`invention uses a non-prescription formulation comprising
`between 0.3-10.0 mg B12 and 0.1-0.4 mg folate, with the
`preferred embodiment using 2.0 mg Bin and 0.4 mg folate.
`Another embodiment of the non-prescription formulation
`uses 0.3-10 mg B12, 0.1-0.4 mg folate, and 5-75 mg B6,
`with the preferred embodiment using 2.0 mg B12, 0.4 mg
`folate, and 25 mg B6. Another embodiment of the present
`invention uses a prescription strength formulation compris-
`ing between 0.3-10.0 mg B12 and 0.4-1.0 mg folate, with
`the preferred embodiment using 2 mg B~2 and 1.0 mg folate.
`In a further embodiment of the present invention, a prescrip-
`tion strength formulation is used comprising 0.3-10 mg B12,
`0.4-1.0 mg folate, and 5-75 mg B~, with the preferred
`embodiment using 2 mg B12, 1.0 mg folate, and 25 mg Br.
`The formulations of the present invention eliminate the
`costly and time-consuming steps of distinguishing between
`vitamin deficiencies once a deficiency is found by measure-
`ment of scram metabolite levels. The present invention is of
`particular benefit to the populations at risk for tissue defi-
`ciencies of Ba~, folate, and B~, such as people over the age
`of 65, and populations that have or are at risk for neurop-
`sychiatric, vascular, renal and hematologic diseases.
`
`BACKGROUND
`
`Vitamins B~:, folate, and B6 are required cofactors in
`metabolic pathways involving metkionine, homocysteine,
`cystathioulne, and cysteine. B12 in the form of 5’-deoxyad-
`enosylcobalamin is an essential cofactor in the enzymatic
`conversion of methylmatonylCoA to succinylCoA. The rem-
`ethylation of homocysteine (HC) to methionine catalyzed by
`methionine synthase requires folate (methyltetrahydro-
`folate) and B~ in the form of methylcobalamin. HC is
`condensed with serine to form cystathionine (CT) in a
`reaction catalyzed by cystathionine ~-synthase which
`requires ]]6 (pyddoxal phosphate). CT is hydrolyzed in
`another Br-dependent reaction to cysteine and ~t-ketobu-
`tyrate.
`It is important to diagnose and treat B~2, folate, and B~
`deficiencies because these deficiencies can lead to life-
`threatening hematologic abnormalities which are completely
`
`2
`reversible by proper treatment. B~ deficiency is a multisys-
`tern disorder with extremely varied clinical presentation
`which has been thought to occur in 0.4% of the population,
`e.g., about 1 million people in the United States. Symptoms
`of B12 deficiency include significant anemia, displayed for
`example in decreased hcmatocrit (e.g., <25%) or hemoglo-
`bin (e.g., -<8 g %), with macrocytic red blood cells (i.e.,
`mean cell volume generally greater than 100 fl), or neuro-
`logic symptoms of peripheral neuropathy and/or ataxia. See,
`for example, Babior and Buan (1983) in Harrison’s Prin-
`ciples of Internal Medicine, (Petersdorf et al., eds.),
`McGraw-Hill Book Co., New York; Lee and Gardner (1984)
`in Textbook of Family Practice, 3rd Ed. (Rakel, ed.), Sann-
`ders & Co., Philadelphia). The hematological abnormalities
`seen are due to intracellular folate deficiency since fo]ate is
`15 required for a number of essential enzymatic reactions
`involved in DNA and RNA synthesis and since the form of
`folate in serum (5-methyltetrahydrofolate) must be metabo-
`lized to tetrahydrofolate by the B12-dependent enzyme
`methionine synthase before it can be utilized by the RNA-
`2o and DNA-related enzymes. While it has been well recog-
`nized that individuals with B~2 deficiency could display
`neurologic disorders in the absence of anemia, such situa-
`tions were believed to be exceptional and rare. See, Beck
`(1985) in Cecil Textbook of Medicine, 17th Ed., (Wyn-
`25 gaarden and Smith, eds.), W. B. Saunders, Philadelphia, pp.
`893-900; Babior and Burro (1987) in Harrison’s Principles
`of lnternal Medicine., !lth Ed., (Braunwald et al., eds.)
`McGraw-Hill, New York, pp. 1498-1504; Walton (1985) in
`Brain’s Diseases of the Nervous System, 9th Ed., Oxford
`3O University Press, Oxford, UK. The neurologic symptoms of
`B~2 deficiency were considered to be late manifestations of
`the disease most typically occurring after the onset of
`anemia or, if they occurred first, were soon to be followed by
`the onset of anemia. See, Woltmarm (1919) Am. J. Med. Sci.
`157:400-409; Victor and Lear (1956) Am. J. Med.
`20:896-911.
`However, it has recently been shown that the textbook
`description of severe megaloblastic anemia and combined
`4o systems disease of the nervous system is the rarest presen-
`tation of B~2 deficiency at the present time (Stabler et al.
`(1990) Blood 76:871-881; Carmel (1988) Arch. Int. Med.
`148:1712-1714; Allen (1991) in Cecil Textbook of Medicine,
`19th Ed., (Wyngaarden and Smith, et al. eds.), W. B.
`45 Saunders, Philadelphia, pp. 846-854.). Therefore, contrary
`to previous teachings, patients that may benefit from B~2
`therapy may have minimal to no hematologic changes while
`manifesting a wide variety of neurologic and psychiatric
`abnormalities (Lindenbaum et al. (1988) N. Engl. J. Med.
`5o 318:1720-1728;~ Greenfield and O’Flynn (1933) Lancet 2:
`62 63). This is particularly true for populations at risk for
`BI~ deficiency, such as the elderly population (Pennypacker
`et al. (1992) J. Am. Geriatric Soe. 40: (in press).
`The incidence of folate deficiency in the population is
`55 unknown, but has been thought to occur commonly in
`individuals with various degrees of alcoholism. The hema-
`tologic abnormalities seen with folate deficiency, such as
`macrocytic anemia, are indistinguishable from those seen
`with Bx2 deficiency. Folate is required for a number of
`60 essential enzymatic reactions involved in DNA and RNA
`synthesis, and is particularly important in rapidly dividing
`cells like those in the bone marrow.
`B6 is required for the first step in heme synthesis and
`serves a major role in transaminatlon reactions of amino acid
`65 metabolism, in decarboxylations, and in the synthesis of the
`neuroactive an’fines histamine, tyramine, serotonin, and
`y-aminobutyric acid (GABA). Clinical manifestations
`
`Sandoz Inc.
`Exhibit 1018-0013
`
`JOINT 1018-0013
`
`

`
`5,563,126
`
`3
`include microcytic hypochromic anemia, characteristic skin
`changes of dermatitis and acrodynia, muscular weakness,
`and a variety of neuropsychiatric abnormalities including
`hyperirritability, epileptiform confusions, depression and
`confusion (Newbeme and Conner (1989) in Clinical Bio-
`chemistry of Domestic Animals, Academic Press, San
`Diego, pp. 796-83z~).
`Vitamin deficiencies are generally determined by mea-
`surement of serum levels. Normal serum B~2 levels are
`200-900 pg/ml, with levels of less than 100 pg/ml being said
`to indicate clinically significant deficiency (Beck (1985)
`supra) However, serum B 12 levels are a relatively insensitive
`determinant of B~2 deficiency in that only 50% of patients
`with clinically confirmed B12 deficiency have levels less
`than 100 pg/ml, 40% are 100-200 pg/ml, and at least 5-10%
`have values in the 200-300 pg/ml range. Diagnosis is further
`complicated by the fact that 2.5% of normal subjects (6,250,
`000 people in the U.S.) have low serum B12 levels (Alien
`(1991) supra), with no evidence of BI~ deficiency and are
`unlikely to benefit from B12 therapy (Schilling et al. (1983)
`Clin. Chem. 29:582; Stabler (1990) supra).
`Normal serum folate levels are 2.5-20 ng/ml, with levels
`less than 2.5 ng/rrd indicating the possibility of clinically
`significant deficiency. Like Bla serum levels, however,
`serum folate levels are a relatively insensitive measure in
`that only 50-75% of patients with folate deficiency have
`levels less than 2.5% ng/ml, with most of the remaining
`25-50% being in the 2.5-5.0 ng/ml range (Allen (1991) in
`Cecil Textbook of Medicine, 19th Ed., supra).
`The development of sensitive serum metabolite assays for
`HC, CT, MMA, and 2-MCA has allowed the relationship
`between metabolite levels and vitamin deficiencies to be
`investigated (Stabler et al. (1987) Anal. Biochem.
`162:185-196; Stabler et al. (1986) J. C!in. Invest.
`77:1606-1612; Stabler et al. (1988) J. Clin. Invest.
`81:466-474). It has been found that elevated serum levels of
`HC and MMA are clinicaily useful tests of functional
`intracellular deficiencies of B ~2 and folate, with elevated HC
`levels seen with both B12 and folate deficiencies, and
`elevated MMA levels seen with aBlz deficiency (Allen et al.
`(1990) Am. J. Hematol. 34:90-98; Lindenbaum et al. (1990)
`Am. J. Hematol. 34:99-107; Lindenbaum et al. (1988) N.
`Engl. J. Med. 318:1720-1728; Beck (1991) in Neuropsy-
`chiatric Consequences of Cobalamin Deficiency, Mosby
`Year Book 36:33-56; Moelby et al. (1990) 228:373-378;
`Ueland and Refsum (19890 J. Lab. CIin. Med.
`114:473-501; Pennypacker et ai. (1992) supra). Increased
`serum levels of CT are seen in both deficiencies and 2-MCA
`is elevated in B ~2 deficiency (Allen et al. (1991) in Proceed-
`ings of the 1st International Congress on Vitamins and
`Biofactors in Life Science, Kobe (Japan); Alien et al. (1993)
`Metabolism (in press)). HC and CT may be elevated in
`patients with intracellular deficiency of Bd, but this has not
`been as well documented (Park and Linkswiler (1970) J.
`Nutr. 100:110-116; Smolin and Benvange (1982) J. Nutr.
`112:1264-1272).
`Elevated serum metabolite levels are observed in disease
`states other than classic vitamin deficiencies. For example,
`elevated HC levels have been observed in the presence of
`vascular disease. The homocysteine theory of atherosclero-
`sis, formulated by McCully and Wilson (1975) Atheroscle-
`rosis 22:215-227, suggests that high levels of HC are
`responsible for the vascular lesions seen in homocystinuria,
`a genetic defect caused by a deficiency in the enzyme
`cystathionine l~-synthase. The theory also implies that mod-
`erate elevations of HC might be associated with increased
`risk for vascular disease (Ueland et al. (1992) in Athero-
`
`4
`sclerotic Cardiovascular Disease, Hemostasis, and Endothe-
`lial Function (Francis, Jr., ed.), Marcel Dekker, Inc., New
`York, pp. 183-236). Moderate hyperhomocysteinaemia has
`been shown to be frequently present in cases of stroke and
`5 to be independent of other stroke risk factors (Brattstrom et
`al. (1992) Eur. J. Clin. Invest. 22:214-221). Clinical and
`experimental evidence demonstrates that patients who are
`homozygotes for cystathionine l~-synthase deficiency have a
`markedly increased incidence of vascular disease and throm-
`bosis. A number of studies (see, Clarke et al. (1991) N. Engl.
`J. Med. 324:1149-1155) strongly suggest that heterozygotes
`for a deficiency of cystathionine I]-synthase also have an
`increased incidence of vascular disease and thrombosis and
`that such heterozygotes may constitute as many as one-third
`of all patients who develop strokes, heart attacks, or periph-
`15 eral vascular disease under age 50. It is also likely that such
`
`10
`
`heterozygotes are also at increased risk for vascular disease
`and thrombosis after age 50. Since the incidence of het-
`erozygosity for cystathionine [3-synthase deficiency is esti-
`mated to be 1 in 60-70, this means that there are approxi-
`20 mately 4 million heterozygotes in the U.S. It is also possible
`
`25
`
`30
`
`that patients with vascular disease due to other causes, such
`as hypercholesterolemia, would also benefit from a decrease
`in their serum HC levels even if their existing levels are only
`slightly elevated or actually within the normal range.
`Renal disease is another condition that gives rise to
`elevated levels of serum metabolites. Approximately 75% of
`patients with renal disease have elevated serum concentra-
`tions of HC, CT, MMA, and 2-MCA. Since patients with
`renal disease have a significant incidence and marked accel-
`eration of vascular disease, it might be beneficial to lower
`their serum metabolite levels, especially that of HC.
`An increasing prevalence of low serum B12 concentra-
`tions with advancing age has been found by many but not all
`35 investigators (Bailey et al. (1980) J. Am. Geriatr. Soc.
`28:276-278; Eisborg et al. (1976) Acta Meal. Scand.
`200:309-314; Niisson-Ehle et al. (1989) Dig. Dis. Sci.
`34:716-723; Norman (1985) 33:374; Hitzhusen et al. (1986)
`Am. J. Clin. Pathol. 85:3236), folate (Magnus et al. (1982)
`40 Scan. J. Haematol. 28:360-366; Blundell et al. (1985) J.
`Clin. Pathol. 38:1179-1184; Elwood et al. (1971) Br. J.
`Haematol. 21:557-563; Garry et al. (1984) J. Am. Geriatr.
`Soc. 32:71926; Hanger et al. (1991) J. Am. Geriatr. Soc.
`39:1155-1159), and B6 (Ranke et al. (1960) J. Gerontol.
`45 15:41-44; Rose et al. (1976) Am. J. Clin. Nutr. 29:847-853;
`Baker et al. (1979) J. Am. Geriatr. Soc. 27:444~450).
`Moreover, prevalence estimates for these vitamin deficien-
`cies vary widely depending on the population groups stud-
`ied. It has been unclear whether this increased prevalence is
`50 a normal age related phenomena or a true reflection of tissue
`vitamin deficiency and whether the low serum vitamin
`doncentrations are a reliable indicator of functional intrac-
`ellular deficiency.
`It is difficult, expensive and time-consuming to distin-
`55 guish between deficiencies of vitamins B~2, folate, and B6.
`The hematologic abnormalities seen with B 12 deficiency are
`indistinguishable from those seen with folate deficiency.
`Similarly to a BI~ deficiency, n6 deficiencies also result in
`hematologic as well as neuropsychiatric abnormalities. The
`60 traditional methods of determining deficiencies by measure-
`ment of serum vitamin levels are often insensitive. As a
`result, in order to determine if and which vitamin deficiency
`is present, a patient will be treated with one vitamin at a time
`and the response to that vitamin determined by normaliza-
`65 tion of serum vitamin levels and the correction of hemato-
`logic abnormalities. These steps are then repeated with each
`vitamin. This method of treatment is both expensive and
`
`Sandoz Inc.
`Exhibit 1018-0014
`
`JOINT 1018-0014
`
`

`
`5,563,126
`
`6
`one or more of the vitamins B~, folate, and Be or for
`neuropsychiatric, vascular, renal, or hematologic diseases.
`One embodiment of the present invention is uses an
`over-the-counter formulation comprised of between 0.3-10
`5 mg CN-cobalamin (Blz) and 0.1-0.4 mg folate. Another
`embodiment of the non-prescription formulation uses
`0.3-10 mg B~2, 0.1-0.4 folate, and 5-75 mg B6. Preferred
`embodiments of the over-the-counter formulation are com-
`prised of about 2.0 mg B~z and 0.4 mg folate, and 2.0 mg
`10 B12, 0.4 mg folate, and 25 mg B6, respectively.
`Another embodiment of the present invention uses a
`prescription formulation comprised of between 0.3-10 mg
`CN-cobalamin (B12) and 0.4-10.0 mg folate. Another
`embodiment of the prescription formulation of the present
`invention uses 0.3-10 mg Ba2, 0.4-1.0 mg folate, and 5-75
`mg B6. Preferred embodiments of the prescription formula-
`tion use about 2.0 mg B12 and 1.0 mg folate, and 2.0 mg B~2,
`1.0 mg folate, and 25 mg B6, respectively.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`15
`
`20
`
`25
`
`FIG. 1 shows the distribution of serum B12 levels for a
`population of elderly outpatients (ages 65-99, n=152) and a
`normal population (ages 17.65, n=100).
`FIG. 2 shows serum MMA levels for a population of
`elderly outpatients with serum B12 values <300 pg/ml (ages
`65-99, n=38/152) and a normal population with serum Bin
`values <300 pg/ml (ages 17-65, n=10/100).
`FIG. 3 shows serum HC levels for a population of elderly
`outpatients with serum Bla values <300 pg/mi (ages 65-99,
`n=38/152) and a normal population with serum B~2 values
`30 <300 pg/rnl (ages 17.65, n=10/100).
`
`35
`
`40
`
`FIG. 4 shows serum MMA levels before and after treat-
`ment with parenteral cobalamin for a population of elderly
`outpatients with elevated MMA values and serum B 12 values
`<300 pg/ml (ages 65-99, n=15/38).
`FIG. 5 shows serum HC levels before and after treatment
`with parenteral cobalan-fin for a population of elderly out-
`patients with elevated HC values and serum Bla values of
`<300 pg/rnl (ages 65-99, n=10/38).
`FIG. 6 shows the distribution of serum Bla levels for a
`population of elderly nursing home patients (ages 55-107,
`n=212) and a normal population (ages 17~5, n=100).
`FIG. 7 shows serum MMA levels for a population of
`elderly nursing home patients with serum Blz values <300
`pg/rnl (ages 55-107, n=29/212) and a normal population
`45 with serum Ba2 values (ages 17-65, n=10/100).
`FIG. 8 shows serum HC levels for a population of elderly
`nursing home patients with serum B~z values <300 pg/rnl
`(ages 55-107, n-29/212) and a normal population with
`serum Ban values <300 pg/ml (ages 17-65, n=10/100).
`FIG. 9 shows serum MMA levels before and after treat-
`ment with parenteral cobalamin for a population of elderly
`nursing home patients with serum B~ values <300 pg/ml
`(ages 55-107, n=14/29).
`
`50
`
`5
`time-consuming. In the presence of multiple deficiencies,
`the diagnosis of vitamin deficiencies is further confused and
`give rise to the dangerous possibility that only one defi-
`ciency will be treated. For example, the hematologic abnor-
`malities seen with a BI~ deficiency will respond to treatment
`with folate alone. However, the neuropsychiatric abnormali-
`ties caused by the BI~ deficiency will not be corrected and
`may indeed by worsened.
`It has now been discovered for the first time that the
`prevalence of intracellular deficiencies of vitamins B12,
`folate, and B6, alone or in combination, is substantially
`higher than that previously estimated by measurement of
`serum vitamin concentrations. The present disclosure estab-
`lishes that tissue deficiencies of one or more of the vitamins
`B12, folate and B6, as demonstrated by the elevated metabo-
`lite concentrations, occurs commonly in the elderly popu-
`lation even when serum vitamin levels are normal. Based on
`this new discovery, the present invention addresses the
`problem of distinguishing between vitamin deficiencies
`when low, low-normal, or normal serum vitamin concentra-
`tions are found by providing formulations for the treatment
`of high serum metabolites and at-risk populations for com-
`binations of one or more tissue deficiencies of vitamins
`folate, and n6.
`Hathcock and Troendle (1991) JAMA 265:96-97, have
`suggested the treatment of pernicious anemia with an oral
`pill containing 300 to 1000 ug or more per day of
`However, contrary to the present invention, Hathcock and
`Troendle teach away from combining B12 therapy with
`folate, since "if the oral cobalamin therapy should fail to
`maintain adequate levels, folate might provide protection
`against development of anemia while permitting nerve dam-
`age from cobalamin deficiency."
`U.S. Pat. No. 4,945,083, issued Jul. 31, 1990 to Jansen,
`entitled: Safe Oral Folic-Acid-Containing Vitamin Prepara-
`tion, describes a oral vitamin preparation comprising
`0.1-1.0 mg Blz and 0.1-1.0 mg folate for the treatment or
`prevention of megaloblastic anemia. This formulation pre-
`sents a problem in the case of a Bla deficient patient, in that
`the 0.5 mg folate may correct the hematologic abnormalities
`present, but the 0.5 mg B~2 dose may be insufficient to
`correct a Bla deficiency due to inadequate intrinsic factor.
`By contrast, the formulation of the present invention teaches
`the use of the combination of B12 and folate, and of BI~,
`folate and B6, sufficient to treat either single or multiple
`deficiencies of B12, folate, and B6. The present invention
`does not rely on the determination of vitamin deficiencies by
`the measurement of serum vitamin levels, but uses the more
`sensitive measurement of elevated serum metabolites of HC,
`CT, MMA, and 2-MCA, shown to be related to the presence
`of BI2 and/or folate and/or to Ba deficiencies or to the
`presence of the increased risk of neuropsychiatxic, vascular,
`renal, and hematologic diseases.
`It is to be understood that both the foregoing general
`description and the following detailed description are exem-
`plary and explanatory only and are not restrictive of the
`invention as claimed.
`
`SUMMARY OF THE INVENTION
`
`This invention includes a method for orally administering
`two new vitamin preparations containing vitamin B~2 and
`folate, and vitamin BI:, folate and B6, for the treatment of
`patients with elevated serum metabolites, such as homocys- 65
`teine, cystathionine, methylmalonic acid, and 2-methylcitric
`acid, as well as populations at risk for tissue deficiencies in
`
`55 FIG. 10 shows serum HC levels before and after treatment
`with parenteral cobalamin for a population of elderly nurs-
`ing home patients with serum Bxz values <300 pg/rnl (ages
`55-107, n=14/29).
`FIG. 11 shows the distribution of serum B~2 levels for a
`6o population of elderly patients (ages 65-99, n=548) and a
`normal population (ages 22-63, n=l17) (Framingham
`study).
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`Reference will now be made in detail to the presently
`preferred embodiments of the invention, which, together
`
`Sandoz Inc.
`Exhibit 1018-0015
`
`JOINT 1018-0015
`
`

`
`5,563,126
`
`7
`with the following examples, serve to explain the principles
`of the invention.
`This invention uses new oral vitamin formulations com-
`bining vitamin B 12 (B ~2, cobalamin) and folic acid (folate),
`and vitamin B12, folate and pyridoxine (Br). The formula-
`tions of the present invention are for use in the treatment of
`elevated serum levels of one or more the metabolites
`homocysteine (HC), cystathionine (CT), methylmalonic
`acid (MMA), or 2-methylcitric acid (2-MCA). The use of the
`formulations of the present invention further include as a
`method of lowering serum metabolites levels of one or more
`of HC, CT, MMA, or 2-MCA, where these metabolite levels
`are not elevated but the patients are at risk for or have
`neuropsychiatric, vascular, renal, or hematologic diseases.
`One embodiment of the present invention uses a non-
`prescription formulation comprised of between about 0.3-10
`mg CN-cobalamin (B12) and 0.1-0.4 mg folate. Another
`embodiment of the present invention uses a non-prescription
`formulation comprised of between about 0.3-10 mg B~2,
`0.1-0.4 mg folate, and 5-75 mg B6. Preferred embodiments
`of the non-prescription formulation are comprised of about
`2.0 mg B!~ and 0.4 mg folate, and 2.0 mg B12, 0.4 mg folate,
`and 25 mg B6, respectively.
`Another embodiment of the present invention is com-
`prised of a prescription formulation comprised of between
`about 0.3-10 mg Blz and 0.4-10.0 mg folate, with the
`preferred embodiment comprised of about 2.0 mg Bt2 and
`1.0 mg folate. Another embodiment of the prescription
`strength formulation is comprised of about 0.3-10 mg B12,
`0.4-10.0 mg folate, and 5-75 mg Br, with a preferred
`embodiment comprised of about 2.0 mg B12, 1.0 mg folate,
`and 25 mg B6.
`The formulations of the present invention are for the
`treatment and prevention of elevated metabolite levels in at
`risk populations, such as the elderly, and people that have or
`are at risk for neuropsychiatdc, vascular, renal and hema-
`tologic diseases. The present invention eliminates the costly
`and time consuming need to differentiate between B12,
`folate, and B6 deficiencies.
`The administration of a daily dose of the vitamin formu-
`lations of the present invention provides better long-term
`normalization of serum HC and other metabolites than prior
`art formulations, and eliminates the difficulty in differenti-
`ating between deficiencies of two or three of the vitamins,
`the difficulty in diagnosing multiple deficiencies of two or
`three of the vitamins, and the expense of doing so. Further,
`the administration of an oral preparation of B12 and folate,
`with or without Br, is preferred over intramuscular injec-
`tions for patient convenience and ease of administration.
`For example, the inclusion of B12 will be useful as a
`safeguard for patients misdiagnosed as folate deficient, even
`though they are actually B~2 deficient, since treatment with
`folate alone in such patients is extremely dangerous. The
`danger arises from the fact that treating a Bz2 deficient
`patient with folate alone may reverse or prevent the hema-
`tologic abnormalities seen in Ba2 deficiency, but will not
`correct the neuropsychiatric abnormalities of a B12 defi-
`ciency and may actually precipitate them. Even in the
`absence of intrinsic factor, approximately 1% of a 2.0 mg
`oral dose of B12 is absorbed by diffusion. Thus, approxi-
`mately 20 ug of B~2 would be absorbed from the formula-
`tions of the present invention which would be more than
`adequate even in patients with pernicious anemia who have
`lost their intrinsic factor-facilitated absorption mechanism
`for B~2. The inclusion of folate will be of benefit since B~2
`deficiency causes a secondary intracellular deficiency of
`
`8
`folate. The inclusion of folate and B6 will also be of benefit
`in patients with mixed vitamin deficiencies.
`The formulations of the present invention may be admin-
`istered as a non-injectable implant