EP0443021A1 - Nouvelle utilisation de composes de vitamine d afin d'inhiber la replication du virus du sida - Google Patents

Nouvelle utilisation de composes de vitamine d afin d'inhiber la replication du virus du sida

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Publication number
EP0443021A1
EP0443021A1 EP90914580A EP90914580A EP0443021A1 EP 0443021 A1 EP0443021 A1 EP 0443021A1 EP 90914580 A EP90914580 A EP 90914580A EP 90914580 A EP90914580 A EP 90914580A EP 0443021 A1 EP0443021 A1 EP 0443021A1
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European Patent Office
Prior art keywords
compound
composition
dihydroxy
vitamin
hydroxy
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EP90914580A
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German (de)
English (en)
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Charles David Pauza
Leonard John Deftos
Hector Floyd Deluca
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Wisconsin Alumni Research Foundation
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Wisconsin Alumni Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to vitamin D compounds and more particularly to the use of vitamin D compounds to treat acquired immune deficiency syndrome
  • AIDS acquired immune deficiency syndrome
  • Vitamin D compounds The effects of Vitamin D compounds on the immune system have only recently been recognized. Rigby,
  • Vitamin D metabolite 1 ⁇ , 25-dihydroxycholecalciferol (1,25DHCC) supports immune function in many in vitro systems.
  • 1,25DHCC a subset of 1,25DHCC that has similar characteristics as a malignant neoplasm originating from many types of immune cells and many of their immunoregulatory cytokines. Rigby, Immunology Today, Vol. 9, pages 54-57, 1988 and Prowedini et al, Bone. Vol. 7, pages 23-28, 1986.
  • One action of 1,25DHCC is to promote the differentiation of monocytes to macrophages and, thus, increase their activity as effector cells of the immune system.
  • Prowedini et al Bone. Vol. 7, pages 23-28, 1986.
  • Other analogs of 1,25DHCC have similar
  • HIV human immunodeficiency virus
  • Vitamin D compounds on the replication of human immunodeficiency virus (HIV) in human cells are described.
  • This action was accompanied by a pro-differentiation effect of the compounds on the phenotype and growth of the cells.
  • the results indicate that these vitamin D compounds as well as others having cellular differentiation activity can be useful in treating the acquired immune deficiency syndrome (AIDS).
  • AIDS acquired immune deficiency syndrome
  • differentiation activity preferably selected from the group consisting of 1 ⁇ -hydroxyvitamin D homolog
  • the treatment may be topical, oral or parenteral.
  • compositions are also disclosed.
  • the compounds are present in the composition in an amount from about 0.01 ⁇ g/gm to about 100 ⁇ g/gm of the
  • composition and may be administered orally or
  • compositions containing one or more side chain unsaturated 1 ⁇ - hydroxyvitamin D homolog compounds for the treatment of human immunodeficiency virus infection and acquired immune deficiency syndrome (AIDS) are provided. Methods employing these compositions are also provided.
  • compositions containing one or more side chain saturated 1 ⁇ -hydroxyvitamin D homolog compounds for the treatment of human immunodeficiency virus infection and acquired immune deficiency syndrome (AIDS) are provided. Methods employing these compositions are also provided.
  • compositions containing one or more 19-nor-vitamin D compounds for the treatment of human immunodeficiency virus and acquired immune deficiency syndrome (AIDS) are provided. Methods employing these compositions are also provided.
  • compositions containing one or more secosterol compounds for the treatment of human immunodeficiency virus and acquired immune deficiency syndrome (AIDS) are provided.
  • the compounds disclosed herein unexpectedly provide highly effective treatments without producing unwanted systemic or local side effects.
  • Fig. 1 is a graph of reverse transcriptase activity of HIV infected U937 cells versus time for three different concentrations of 1,25-dihydroxyvitamin D 3 (1,25 DHCC);
  • Fig. 2 is a graph of percent inhibition of
  • Fig. 3 is a graph of percent inhibition of HIV replication in U937 cells and percent cellular differentiation versus concentrations for three different vitamin D compounds;
  • Fig. 4 illustrates hybridization analysis of viral RNA in vitamin D 3 treated U937 cells;
  • Fig. 5 is a graph of percent cellular
  • Fig. 6 illustrates various structures of the vitamin D metabolites and analogs tested.
  • Fig. 7 illustrates the ability of OH analogs to induce HL-60 cell differentiation as assayed by nitroblue tetrazolium reduction.
  • Fig. 8 illustrates the induction of phagocytic activity in HL-60 cells by a series of 25-OH-D 3 and 25-OH-D 2 metabolites and analogs.
  • Fig. 9 illustrates the effect of side chain elongation and/or truncation and 5, 6-isomerization on the ability of the 1,25-(OH) 2 D 3 analog to induce nonspecific acid esterase activity in HL-60 cells.
  • Fig. 10 illustrates the activity of short chain and primary alcohol analogs of 1,25-(OH) 2 D 3 in inducing NBT reducing activity in HL-60 cells after a 4-day incubation.
  • Fig. 11 illustrates the nonspecific acid esterase activity induced by 1,24R,25-(OH) 3 D 3 and 1,25-(OH) 2 D 2 in HL-60 cells.
  • Vitamin D 3 Myelodysplastic syndromes. Uchino et al, eds, Elsevier, pp. 133-138, 1988) were evaluated for their effect on HIV replication in infected U937 cell cultures.
  • Figure 1 illustrates that 1,25-dihydroxycholecalciferol (1,25DHCC) inhibits HIV-1 replication in U937 cells.
  • Open circles correspond to 10 -10 M, filled triangles to
  • 1,25DHCC inhibited viral replication at all doses tested. The effect was dose dependent and curves representing the time course of virus production shifted to the right (Figurr 1). At 10- 9 M drug concentration, the peak of virus production was observed on day 3 , compared to the peak on day 2 in untreated cultures and was reduced by approximately 3-fold. Little evidence for HIV production was observed in the 10 -9 M samples. At 10 -10 M virus replication became restricted on day 2, similar to control cultures, although the magnitude of this peak was reduced sharply. Accordingly, the dose-response relationship between 1,25DHCC treatment and HIV replication in U937 cells revealed two independent activities of this agent: a quantitative inhibition of virus growth and a qualitative change in the pattern of virus replication. Both of these features attest to the ability of 1,25DHCC to inhibit acute HIV infection of U937 cells.
  • day 2 post-infection was used to compare the effects of Vitamin D compounds on HIV replication in U937 cells.
  • Cells were infected in batches for 2.5 hours and then washed and distributed to individual flasks containing the appropriate
  • Figure 2 illustrates a comparison of the dose-response curves for six analogues of Vitamin D 3 , tested for their ability to inhibit HIV replication in U937 cells. In this case, samples were collected at day 2 post-infection. HIV infection and reverse
  • transcriptase assays were as described in Fig. 1.
  • Figure 2 shows that two 1,25DHCC analogues, namely, 1,25DHDFCC and 1,25DHHDCC, inhibited HIV
  • Figure 3 illustrates the effects of 1,25-dihydroxycholecalciferol (1,25DHCC), 1,25-dihydroxyhexadeuterocholecalciferol (1,25DHHDCC) and 1,25-dihydroxydifluorocholecalciferol (1,25DHDFCC) on U937 differentiation and HIV replication.
  • Figure 3 illustrates the effect of these metabolites on two measures of cellular differentiation, i.e., the
  • the filled circles designate the capacity of these compounds to inhibit HIV replication as assessed by cellfree reverse transcriptase activity on day 2 postinfection.
  • the filled squares show the percentage of cells positive for tetrazolium reductase activity as evaluated by in situ cytologic assay (See Prowedini et al, Bone, Vol. 7 pages 23-28, 1986).
  • 1,25-DHDFCC 4.7 ⁇ 10 -9 M 7.6 ⁇ 10 -10 M 1.2 ⁇ 10 -9 M +Differentiation was measured as the acquisition of capacity to reduce tetrazolium dye in situ (as described in Prowedini et al, Bone, Vol. 7, pages 23-28, 1986).
  • the half-maximal concentration is determined to be that concentration of drug able to induce 50% of the
  • Figure 4 illustrates hybridization analysis of viral RNA in Vitamin D 3 -treated U937 cells. Infections were performed as described with respect to Fig. 1. Time course analyses showed that the peak of viral RNA production occurred on day 2 post-infection; accordingly, this sampling interval was chosen.
  • Total cellular RNA was prepared by guanidinium-isothiocyanate extraction and gradient purification. The purified samples were dissolved in water and the concentration of nucleic acid was determined from the optical density at 260 nm. Twenty micrograms of each RNA was loaded per lane. The 9.2, 4.3 and 2.0 kb HIV RNA species were detected by hybridization with a LTR-specific fragment, and their positions are indicated on Figure 4. After autoradiographic exposure to reveal the pattern of HIV-1 gene expression, the filters were stripped and
  • ⁇ -actin mRNA serves as a control.
  • Vitamin D compounds thus tested produced a decrease in the HIV ribonucleic acid (RNA) of the treated cells, i.e. the intensity of hybridization in the Vitamin D-treated cultures was approximately one-half that observed in the untreated control culture. This value is consistent with the decreased virus production demonstrated in Figure 1.
  • vitamin D compounds useful in the compositions of the present invention and for the treatment of acquired immune deficiency syndrome (AIDS) are those which induce
  • vitamin D compounds defined by the above functions are those selected from the group consisting of 1 ⁇ -hydroxyvitamin D homolog compounds, 19-nor vitamin D compounds and secosterol compounds.
  • the 1 ⁇ -hydroxyvitamin D homolog compounds useful in the present invention are characterized
  • R 4 and R 5 represent hydrogen, deuterium, flourine or when taken together R 4 and R 5 represent a carboncarbon double bond or a carbon-carbon triple bond
  • R 13 represents hydrogen, deuterium, hydroxy, protected hydroxy, fluorine or an alkyl group
  • Z represents
  • R 3 represents hydrogen, hydroxy, protected hydroxy, fluorine or an alkyl group
  • X and Y which may be the same or different are hydrogen or a hydroxy-protecting group
  • R. represents the group -CF 3 , -CD 3 , or -(CH 2 ) q -H
  • R 2 represents the group -CF 3 , -CD 3 , or -(CH 2 ) p -H
  • n, q and p are integers having independently the values of 1 to 5
  • R 1 and R 2 when taken together represent the group -(CH 2 ) m -where m is an integer having the value of 2 to 5.
  • the 19-nor-vitamin D compounds referred to herein are a class of 1 ⁇ -hydroxylated vitamin D compounds in which the ring A exocyclic methylene group (carbon 19) typical of all vitamin D systems has been removed and replaced by two hydrogen atoms. Structurally these novel analogs are characterized by the general formula II shown below:
  • U may be an alkyl, hydrogen, hydroxyalkyl or fluoroalkyl group, or U may represent the following side chain:
  • Z 1 represents hydrogen, hydroxy or O-acyl
  • R 6 and R 7 are each selected from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl, deuteroalkyl or, when taken together represent the group — (CH 2 )m — where m is an integer having a value of from 2 to 5,
  • R 8 is selected from the group consisting of hydrogen, deuterium, hydroxy, fluorine, O-acyl, alkyl, hydroxyalkyl and fluoroalkyl
  • R 9 is selected from the group consisting of hydrogen, deuterium, fluorine, alkyl, hydroxyalkyl and fluoroalkyl, or, R 8 and R 9 taken together represent double-bonded oxygen or double-bonded carbon
  • R 10 and R 11 are each selected from the group consisting of hydrogen, deuterium, hydroxy, O-acyl, fluorine and alkyl, or, R 10 and R 11 taken together form a carbon-carbon double bond or a carbon-carbon triple bond
  • side chains for the 19-nor compounds are the structures represented by formulas (a), (b), (c), (d) and (e) below, i.e. the side chain as it occurs in 25-hydroxyvitamin D 3 (a);
  • vitamin D 3 (b); 25-hydroxyvitamin D 2 (c); vitamin D 2 (d); and the C-24-epimer of 25-hydroxyvitamin D 2 (e).
  • the class of secosterol compounds referred to herein has a similarity with some of the known vitamin D compounds. Unlike the known vitamin D compounds, however, the secosterols used in the present invention do not express the classic vitamin D activities in vivo, i.e. stimulation of intestinal calcium transport, or the mobilization of bone calcium, and hence they cannot be classified as vitamin D
  • R 12 is hydrogen, methyl, ethyl or propyl and where each of X 2 and Y 2 represent, independently, hydrogen, an acyl group, or a hydroxy-protecting group.
  • hydroxy-protecting group refers to any group commonly used for the protection of hydroxy
  • a "protectedhydroxy” is a hydroxy function derivatized by one of the above hydroxy-protecting groupings.
  • Alkyl represents a straight-chain or branched hydrocarbon radical of 1 to 10 carbons in all its isomeric forms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, etc., and the terms "hydroxyalkyl” and “fluoroalkyl” refer to such an alkyl radical substituted by one or more hydroxy or fluoro groups respectively.
  • An acyl group is an alkanoyl group of 1 to 6 carbons in all its isomeric forms, or an aroyl group, such as benzoyl, or halo-, nitro- or alkylsubstituted benzoyl groups, or a dicarboxylic acyl group such as oxalyl, malonyl, succinoyl, glutaroyl, or
  • aryl signifies a phenyl-, cr an alkyl-, nitro- or halo-substituted phenyl group.
  • the term “24-dihomo” refers to the addition of two methylene groups at the carbon 24 position in the side chain
  • trihomo refers to the addition of three methylene groups at the same position so that both additions have the effect of extending the length of the side chain.
  • the term “26,27-dimethyl” refers to the addition of a methyl group at the carbon 26 and 27 positions so that for example R 1 and R 2 are ethyl groups.
  • the term “26,27-diethyl” refers to the addition of an ethyl group at the 26 and 27 positions so that R 1 and R 2 are propyl groups.
  • R 4 and R 5 represent a double bond
  • R 4 and R 5 represent a double bond
  • R 4 and R 5 represent a double bond
  • these compounds when the side chain is unsaturated are: 24-dihomo-1,25-dihydroxy-22-dehydrovitamin D 3 , i.e. the compound shown above, where X and Y are hydrogen, Z is hydroxy, n equals 3, and R 1 and R 2 are each a methyl group; 26,27-dimethyl-24-dihomo-1,25-dihydroxy-22-dehydrovitamin D 3 , i.e.
  • R 4 and R 5 each represent hydrogen
  • R 4 and R 5 each represent hydrogen
  • R 4 and R 5 are: 24-dihomo-1,25-dihydroxy-vitamin D 3 , i.e. the compound shown above, where X and Y are hydrogen, Z is hydroxy, n equals 3, and R 1 and R 2 are each a methyl group; 26,27-dimethyl-24-dihomo-1,25-dihydroxy-vitamin D 3 , i.e. the compound shown above where X and Y are hydrogen, Z is hydroxy, n equals 3, and R 1 and R 2 are each an ethyl group; 24-trihomo-1, 25-dihydroxy-vitamin D 3 , i.e.
  • Suitable starting materials are, for example, the vitamin D compounds of the general structure IV:
  • vitamin D starting materials are known compounds, or compounds that can be prepared by known methods.
  • the hydroxy group is converted to the corresponding acyl derivative, i.e. the compound V shown above, where X 3 represents an acyl group, using standard acylation procedures, such as treatment with an acyl anhydride or acyl halide in pyridine at room
  • the next step of the process comprises the reduction of the 10-oxo-group to the corresponding 10- alcohol having the structure VIII shown below (where X 3 is acyl and Y 3 represents hydroxy).
  • X 3 is acyl
  • this reduction is carried out conveniently in an organic solvent at from about 0°C to about room temperature, using NaBH 4 or equivalent hydride reducing agents, selective for the reduction of carbonyl groups without cleaving ester functions.
  • any of the other hydride reducing agents e.g. LiAlH 4 , or analogous reagents
  • any of the other hydride reducing agents e.g. LiAlH 4 , or analogous reagents
  • the 10-hydroxy intermediate is then treated with an alkyl-or arylsulfonylhalide (e.g.
  • a 1-O-acyl function in the precursor compound VII is also cleaved in this reduction step to produce the free 1 ⁇ -hydroxy function, and any O-acyl protecting group in the side chain would, of course, likewise be reduced to the corresponding free alcohol function, as is well understood in the art.
  • the hydroxy groups at C-1 can be
  • a 1-O-acyl function in the precursor compound VII is also cleaved in this reduction step to produce the free 1 ⁇ -hydroxy function, and any O-acyl protecting group in the side chain would, of course, likewise be reduced to the corresponding free alcohol function, as is well understood in the art.
  • the hydroxy groups at C-1 can be
  • structure IX or X or the free 1,3-dihydroxy compound can, of course, be reacylated according to standard procedures with any desired acyl group, so as to produce the product of structure II above, where X 1 and Y 1 represent acyl groups which may be the same or different.
  • X 1 and Y 1 represent acyl groups which may be the same or different.
  • the 19-nor-vitamin D compounds useful in this invention are more specifically described by the
  • cleavage reaction does not require elevated temperatures, and it is, indeed, generally preferable to conduct the reaction at approximately room temperature,
  • IXa and Xa were hydrolyzed in the same manner.
  • Each of the monoacetates was dissolved in 0.5 ml of ether and 0.5 ml 0.1 N KOH in methanol was added. The mixture was stirred under argon atmosphere for 2 h. More ether was added and the organic phase washed with brine, dried over anhydrous MgSO 4 , filtered and evaporated. The residue was dissolved in a 1:1 mixture of 2-propanol and hexane and passed through a Sep Pak column and washed with the same solvent. The solvents were evaporated and the residue purified by HPLC (Zorbax Sil, 6.4 x 25 cm, 10% 2-propanol in hexane).
  • the secosterol of structure III where R 12 is hydrogen can be prepared according to the method of Lam et al as published in Steroids 26, 422 (1975), the description of which is specifically incorporated herein by reference.
  • the secosterols of structure III, where R 12 is methyl, ethyl or propyl can be prepared according to the general process illustrated and described in U. S. Patent No. 4,800,198 issued January 24, 1989 entitled “Method of Inducing the Differentiation of Malignant Cells With Secosterol", the description of which is specifically incorporated herein by reference.
  • compounds are one or more side chain unsaturated or side chain saturated 1 ⁇ -hydroxyvitamin D homolog compound, one or more 19-nor-vitamin D compound, or one or more
  • composition 10.0 ⁇ g per gm of composition, and may be administered topically, orally or parenterally in dosages of from about 0.1 ⁇ g/day to about 100 ⁇ g/day.
  • a concentration of 0.01 ⁇ g per gm of the composition is preferred.
  • the compounds may be formulated as creams, lotions, ointments, topical patches, pills, capsules or tablets, or in liquid form as solutions, emulsions, dispersions or suspensions in pharmaceutically innocuous and acceptable solvent or oils, and such preparations may contain in addition other pharmaceutically innocuous or beneficial components, such as antioxidants, emulsifiers, coloring agents, binders or coating materials.
  • the compounds may be administered topically, as oral doses, or parenterally by injection or infusion of suitable sterile solutions.
  • the compounds are advantageously administered in amounts sufficient to effect the differentiation of Promyelocytes to normal macrophages. Dosages as described above are suitable, it being understood that the amounts given are to be
  • NSE nonspecific acid esterase
  • NBT nitroblue tetrazolium
  • Test Results Data is presented in Table 2 as the percent of differentiated cells resulting from treatment with various concentrations of 1,25-(OH) 2 D 3 (used as comparison standard) or one of the four
  • Results arc expressed as percent of total cells counted that have differentiated.
  • Vitamin D deficient rats were fed a low-calcium diet and given the indicated daily dose of compound in propylene glycol intraperitoneally or by Alzet minipump (experiment I) for 7 days. Controls received the vehicle. At 7 days the rats were killed for the determinations. There were at least six rats per group. Statistical analysis was done by Student's t test.
  • serum Ca b 3 from a, p ⁇ 0.01; b 2 from a, NS.
  • serum Ca b 1 , b 2 from a, p ⁇ 0.005; c 2-4 , d 1-5 from a, NS; b 1 , b 2 from c 2-4 , p ⁇ 0.001; b 1 , b 2 from d 1-5 , p ⁇ 0.001; d 1-5 from a 1 and a, NS.
  • serum Ca b from a, p ⁇ 0.001; c 1-4 from a, NS; b from c 1-4 , p ⁇ 0.001.
  • 24,24-dihomo-1,25-(OH) 2 D 3 and 24-homo-1,25-(OH) 2 D 3 are approximately 10 times more active than the native hormone in causing differentiation of HL-60 cells.
  • the addition of more than one carbon at the carbon 24 position does not increase differentiative activity further.
  • the addition of an additional carbon at the carbon 24 position as in ⁇ 22 -24,24,24-trihomo-1,25-(OH) 2 D 3 results in
  • the intestinal calcium transport assay represented by Table 3, for example, shows the known active metabolite, 1,25-(OH) 2 D 3 to
  • the 24-homo-1,25-(OH) 2 D 3 compound is less active than 1,25-(OH) 2 D 3 in this test.
  • the dihomo compounds do not saturate even when provided at 285 pmol and thus are at least 10 times less active than 1,25-(OH) 2 D 3 .
  • the trihomo compound shows little or no activity at even 1096 pmol/day. Although exact estimates of activity in this system are not possible from the data available, it is clear that the dihomo and trihomo compounds are at least 10 times less active in intestinal calcium transport than is 1,25-(OH) 2 D 3 .
  • the 24,24-dihomo-1,25-(OH) 2 D 3 compounds whether saturated in the 22-position or unsaturated, have 10-fold higher HL-60 differentiative activity than 1,25- (OH) 2 D 3 but have markedly diminished calcium mobilizing activity.
  • the 24-homo-1,25-(OH) 2 D 3 shows a 10-fold increase in the HL-60 activity and a 5-10 fold decrease in calcium mobilizing activity. If the differentiative activity is of thereapeutic importance in the treatment of AIDS as the data presented herein indicates, then the 24-homologated 1,25-(OH) 2 D 3 compounds may be very
  • the 19-nor compounds of this invention also exhibit a pattern of biological activity similar to the above homologated compounds, namely, high potency in promoting the differentiation of malignant cells and little or no activity in calcifying bone tissue. This is illustrated by the biological assay results obtained for 1 ⁇ ,25-dihydroxy-19-nor-vitamin D 3 which are summarized in Tables 4 and 5, respectively.
  • Table 4 shows a comparison of the activity of the known active metabolite 1 ⁇ ,25-dihydroxyvitamin D 3 and the 19-nor analog 1 ⁇ ,25-dihydroxy-19-nor-vitamin D 3 in inducing the
  • test compounds were conducted according to known procedures, as given, for example, by DeLuca et al. (U.S. Patent 4,717,721 and Ostrem et al., J. Biol. Chem. 262, 14164, 1987).
  • the differentiation activity of the test compounds is expressed in terms of the percent of HL-60 cells having differentiated to normal cells in response to a given concentration of test compound.
  • the 19-nor analog exhibits no activity in an assay measuring the calcification of bone, a typical response elicited by vitamin D compounds.
  • Relevant data, representing the results of an assay comparing the bone calcification activity in rats of 1 ⁇ , 25-dihydroxyvitamin D 3 and 1 ⁇ ,25-dihydroxy-19-nor vitamin D 3 are summarized in Table 5. This assay was conducted according to the procedure described by Tanaka et al., Endocrinology 92, 417 (1973).
  • Each assay group comprised 6 rats, receiving the indicated amount of test compound by intraperitoncal injection daily for a period of seven days.
  • the 19-nor analog shows a selective activity profile combining high potency in inducing the differentiation of malignant cells with very low or no bone calcification activity.
  • the compounds of this novel structural class therefore, can be useful as therapeutic agents for the treatment of AIDS.
  • the table above also includes the cell differentiation activity exhibited by 1 ⁇ -hydroxyvitamin D 3 (1 ⁇ -OH-D 3 ) and 1 ⁇ ,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), two known vitamin D derivatives with potent antileukemic action.
  • the tabulated data show that the level of activity of the seco sterols is lower than that shown by 1,25-(OH) 2 D 3 (the most potent vitamin D-derived agent for differentiation of leukemia cells), but is approximately equivalent to that shown by 1 ⁇ -hydroxyvitamin D 3 , a compound known to be effective in the treatment of human leukemoid diseases (Suda et al., U.S.
  • the compound does not elevate serum calcium levels and thus is devoid of significant bone calcium mobilization activity.
  • the compound does not stimulate calcium transport in the intestine at a dose level of 6250 picomole per animal.
  • the secosteroids of this invention provide a novel and preferred method for the treatment of viral diseases such as AIDS. Bone calcium mobilization activity of 1 ⁇ ,25-(OH) 2 -26-homo-D 3 compounds
  • Serum calcium concentration was determined with an atomic absorption spectrophotometer
  • 1 ⁇ ,25-(OH) 2 D 3 the most potent vitamin-derived antileukemic agent known thus far, is also the most potent calcemic agent, and the antileukemic potency of 1 ⁇ -OH-D 3 is likewise correlated with high calcemic activity.
  • the administration of these compounds, at the dosage level where they are effective as antileukemic drugs would necessarily produce elevated, potentially excessive, calcium levels with attendant serious medical complications,
  • the compounds of this invention are also preferentially active in inducing the differentiation of malignant cells to non-malignant cells, i.e. in antineoplastic activity as measured by leukemia cell differentiation, while being no more active than 1 ⁇ , 25-dihydroxyvitamin D 3 in their effect on calcium metabolism. Because of this unique and unexpected combination of properties, the novel side-chain homovitamin D compounds of this invention represent superior and preferred agents for the trea ment of leukemias, and viral diseases, such as AIDS.
  • the side-chain homovitamin D compounds of this invention When administered to human promyelocytic leukemia cells (HL-60 cells) grown in culture, the side-chain homovitamin D compounds of this invention induce the differentiation of these cells to macrophages (monocytes). In several standard assays for measuring differentiation activity, these compounds were shown to be more effective than 1 ⁇ ,25-(OH) 2 D 3 , the most active vitamin D derivative known thus far.
  • the extent of differentiation induced by the tested vitamin D derivatives was expressed as the percentage of cells that exhibit functional and enzymatic markers
  • the two markers assayed were a) the ability of the cells to phagocytize dead yeast, and b) the ability of the cells to produce superoxide (reduce nitrotetrazalium blue) when stimulated with phorbol esters.
  • % phagocytic cells indicates the percent of differentiation induced by the test compounds.
  • both of the above assays confirm the high potency of the homovitamin D compounds in inducing the differentiation of leukemic cells.
  • the above results show that in this differentiation activity these homovitamin D compounds are significantly more potent than 1 ⁇ ,25-(OH) 2 D 3 .
  • the homovitamin D compounds of this invention are:
  • Suitable dosage amounts are as described above, it being understood that dosages can be adjusted according to the severity of the disease or the response or the condition of subject as is well-understood in the art.
  • Biological Activity of Cyclopentano Vitamin D Analogs are as described above, it being understood that dosages can be adjusted according to the severity of the disease or the response or the condition of subject as is well-understood in the art.
  • vitamin D analogs cyclopentano-1,25-dihydroxy-vitamin D 3 and cyclopentano-1,25-dihydroxy-22E-dehydro-vitamin D 3 were assayed for both calcemic
  • Sprague Dawley Co., Madison, WI were fed a low calcium, vitamin D-deficient diet (0.22% Ca, 0.3% P) as described by Suda et al. (J. Nutr. 100, 1049-1052, 1970), for a total of 4 weeks ad libitum.
  • the animals were divided randomly into groups of 6 rats each.
  • One group (the control group) received a daily dose of solvent vehicle (0.1 mL of 95% propylene glycol/5% ethanol) by interperitoneal (i.p.)
  • test compound i.e. 1,25-(OH) 2 D 3 , compound I, or compound II
  • Table 11 the amounts of test compound dissolved in the same amount of solvent vehicle by daily injection over a period of 7 days.
  • the animals were killed 24 hours after the last
  • Intestinal calcium transport was measured by the everted gut sac technique [Martin & DeLuca, Am. J. Physiol. 216, 1351 (1969)] as described by Halloran and DeLuca [Arch.
  • neoplastic disease especially leukemias
  • 1,25-Dihydroxyvitamin D 3 the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro.
  • the relative activity of 30 analogs of 1,25-dihydroxyvitamin D 3 in inducing development of monocytic markers in HL-60 cells was assessed.
  • the three differentiation markers assayed were nonspecific acid esterase activity, nitroblue tetrazolium reducing activity, and phagocytic capacity.
  • AR Relative activity
  • Figure 6 shows the structures of most of the analogs studied.
  • Figures 7 through 11 are representative log dose-response curves for the various analogs. Steroidal side-chain structures are shown above each curve, and each figure provides the curves for one of the assays used. Similar curves were prepared for the other 2 assays and were used to calculate the ED 50 s for each analog. Table 13 provides the ED 50 s determined by each of the 3 assays for most of the analogs as well as the calculated activity ratios (AR).
  • AR activity ratios
  • Figure 7 shows the activity of five 1 ⁇ -hydroxylated analogs that do not contain side-chain hydroxyl groups.
  • methylation of the 25-hydroxy group to produce the methyl ether of the natural hormone (In) results in only a 6-7-fold decrease in activity.
  • Introduction of a ⁇ 22 -trans double bond improves the activity of
  • 25-OH-D 2 (2b) and 24-epi-25-OH-D 2 (2c) can induce 50% differentiation at 4 x 10 -7 M and 3 x 10 -7 M, respectively, being approximately 2-fold more active than 25-OH-D 3 .
  • This observation agrees with the result in Figure 7 that introduction of a trans double bond at C-22 improves the activity 2-fold.
  • Both 25-OH-D 2 isomers have approximately the same activity in this system indicating a tolerance for either R- or S-methyl stereochemistry at C-24.
  • Introduction of fluorine groups in the side chain as in 26,27-F 6 -25-OH-D 2 (2e) improves the activity of 25-OH-D 2 two-fold. This agrees with previous observations of Shiina et al. and Koeffler et al. that fluorination either in the 24-pos ⁇ tion or 26,27-position improves ability of 1,25-(OH) 2 D 3 to induce myeloid cell maturation 4 to 7-fold.
  • Figure 9 Generally, elongation by one carbon improves the activity of the natural hormone by one order of magnitude, while truncation of the side-chain by each carbon removed diminishes activity by one order of magnitude.
  • 24-Homo-1,25-(OH) 2 D 3 (1b) and 26-homo-1,25-(OH) 2 D 3 (1c) are 8-fold more active than the natural hormone since they can induce 50Z maturation of the HL-60 cells at 1.3 x 10 -9 M compared to the 10 -8 M required for 1,25-(OH) 2 D 3 .
  • Introduction of unsaturation at C-22 resulted in analogs that retained the 8-fold improved potency
  • a derivative with a 5,6-trans-triene modification is only 7 times less effective than the 5,6-cis compound ( Figure 9).
  • 7 ⁇ 10 -7 M concentration of the 5,6-trans isomer of 24-nor-1,25-(OH) 2 D 3 is required to achieve 50% differentiation compared to 1.3 ⁇ 10 -7 M needed of the 5,6-cis derivative (1d).
  • Figure 10 presents the activity of primary alcohol side chains of various lengths as well as short chain analogs (compounds 1o-1v).
  • 26,27-bis-nor-1,25- (OH) 2 D 3 (1o) differs from the natural hormone only in its lack of the two methyl groups flanking the 25-hydroxy substituent. Yet, this compound is two orders of magnitude less effective than 1,25-(OH) 2 D 3 .
  • Sequential deletion of one carbon from the side chain of 1o represented by analogs 1p-1r has no further effect in decreasing the activity: 23,24,25,26,27-pentanor-1,22-(OH) 2 D 3 (1r) is also two orders of magnitude less effective than 1,25-(OH) 2 D 3 .
  • 1,25-(OH) 2 D 3 1,25-(OH) 2 D 3 .
  • 1 ⁇ -OH-D 3 an analog that has lost the 25-hydroxyl substituent while retaining the original length of the steroidal side chain is 100-fold less active than 1,25-(OH) 2 D 3 , these results are remarkable.
  • the high activity of 1t and 1u can be explained in terms of their surprising high affinity for the 1,25-(OH) 2 D 3 receptor.
  • 1 ⁇ -OH-homopregnacholecalciferol and 1 ⁇ -OH-bishomopregnacholecalciferol are only 4- and 11-fold less effective than 1,25-(OH) 2 D 3 in displacing the natural ligand from its binding site on the chick intestinal receptor (22).
  • 1,24R-(OH) 2 D 3 (1f) has equivalent activity with the natural hormone, while its stereoisomer, 1,24S-(OH) 2 D 3 (1g) is half as active (Table 13). In vivo, 1,24S-(OH) 2 D 3 is less active in stimulating calcium transport and bone calcium mobilization and has equal affinity for the receptor compared to
  • 1,25-(OH) 2 D 3 Matsui et al. have also shown that 1,24R-(OH) 2 D 3 shows the same potency as 1,25-(OH) 2 D 3 in inducing monocyte/granulocyte associated plasma membrane antigens, and 1,24S-(OH) 2 D 3 is only slightly less active. This
  • 1,25-(OH) 2 D 3 (Table 13).
  • 1,25-(OH) 2 D 3 (1i) and 1,24R,25-(OH) 3 D 3 (1h) uniquely produce unexpected and highly reproducible biphasic log dose-response curves (Figure 11) with 2 ED 50 s at 2-3 x 10 -8 M and 9 x 10 -8 M.
  • 1,25-dihydroxyvitamin D 3 is the most active; fifty percent of the cells exhibit the mature phenotype following a 4-day treatment with 10 -8 M 1,25-dihydroxyvitamin D 3 . Removal of either the C-1 or C-25-hydroxyl group reduces activity by two orders of
  • Analogs with short aliphatic side chains as 1 ⁇ -hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone.
  • the activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D 3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D 3 is receptormediated.
  • 1 ⁇ hydroxyvitamin D 3 is less than 100 times as active as 1 ⁇ ,25-dihydroxyvitamin D 3 (see Table 13) in causing differentiation of HL60 cells in vitro. However, in vivo it is well established that 1 ⁇ -hydroxyvitamin D 3 is rapidly converted to 1 ⁇ ,25-dihydroxyvitamin D 3 , Hollick et al, Science, Vol. 190, pages 576-578 (1975) and
  • compositions and methods for treating lentivirus are provided.
  • lentiviruses are well known, and in general terms can be described as retro viruses having a relatively slow pathology with a genetic structure common to this group of viruses.
  • lentiviruses include human immunodeficiency virus type 1 (HIV-1), human
  • HIV-2 immunodeficiency virus type 2
  • simian simian
  • BLV leukosis virus
  • FMV feline immunodeficiency virus
  • HL-60 cells were cultured for four days in the presence of the indicated concentration of 1,25-(OH) 2 D 3 analogs.
  • the analog concentration capable of inducing 50% maturation by the three assays was derived from log dose-response curves.
  • AR is the ratio of the analog average ED 50 to the ED 50 for

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Abstract

Nouvelle utilisation du composé 1alpha, 25-dihydroxycholécalciférol de la vitamine D et de ses analogues apparentés et dérivés, afin de traiter le syndrome d'immunodéficience acquise humain, SIDA, dans la mesure ou ces agents inhibent la réplication dans les cellules humaines du VIH, le virus d'immunodéficience humaine provoquant le syndrome d'immonodéficience acquise, SIDA.
EP90914580A 1989-09-11 1990-09-10 Nouvelle utilisation de composes de vitamine d afin d'inhiber la replication du virus du sida Withdrawn EP0443021A1 (fr)

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EP0998290B1 (fr) * 1997-06-06 2004-09-15 Wisconsin Alumni Research Foundation Utilisation de composes de la vitamine d pour eviter les rejets de greffes
WO2001064211A1 (fr) * 2000-03-03 2001-09-07 Lee Shu Wen Médicament spécifique pour le traitement du sida
US7491712B1 (en) 2007-12-10 2009-02-17 Formosa Laboratories, Inc. Process for preparation of paricalcitol and intermediates thereof

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US4225596A (en) * 1978-10-13 1980-09-30 Wisconsin Alumni Research Foundation Method for treating calcium imbalance and improving calcium absorption in mammals
FR2530624B1 (fr) * 1982-07-26 1987-08-07 Wisconsin Alumni Res Found Nouveaux analogues de la vitamine d et compositions pharmaceutiques ou veterinaires les contenant
US4728643A (en) * 1984-11-02 1988-03-01 The General Hospital Corporation Method of treating psoriasis
DE3680740D1 (de) * 1985-04-23 1991-09-12 Wisconsin Alumni Res Found Die differenzierung maligner zellen bewirkende seco-sterol-verbindungen.
US4749710A (en) * 1985-05-01 1988-06-07 Hoffmann-La Roche Inc. Immunosuppressive agents

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