26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3

A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.     

Determination of biological activity of 24,24-difluoro-1,25-dihydroxyvitamin D3 in the chick using a new method for assessing intestinal calcium uptake

Oxygen-dependent calcium uptake by chick duodenal discs has been characterized and used to assay the relative activities of 1,25-dihydroxyvitamin D₃ and its 24,24-difluoro analog. The calcium uptake was found to be stimulated by low sodium (30 mm) and phosphate (0.01–0.3 mm). The rate of oxygen-dependent calcium uptake was found half-maximal at a calcium concentration of 5 mm. At a concentration of 5 mm calcium, the uptake was linear for at least 15 min with approximately a threefold stimulation by prior administration of 1,25-dihydroxyvitamin D₃ (125 ng). This determination, as well as increase in serum calcium and percentage bone ash, was used to assess the biological activities of 1,25-dihydroxyvitamin D₃ and its 24,24-difluoro analog. The difluoro analog is about four to five times more active than 1,25-dihydroxyvitamin D₃ as measured in each of these systems.     

Studies on the mode of action of calciferol: Comparison of the biochemical properties of an antiserum and the chick intestinal receptor both specific for 1,25-dihydroxyvitamin D3

The structural requirements for the interaction of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] with an anti-1,25(OH)₂D₃ antiserum and with the natural cytosolic receptor for 1,25(OH)₂D₃ isolated from chick intestine have been evaluated quantitatively. The antiserum was raised in a rabbit against a 1,25(OH)₂D₃-hemisuccinate derivative which was linked to bovine serum albumin at the C-3 position of the steroid. For these cross-reaction studies structural analogs of 1,25(OH)₂D₃ were used in competitive protein binding assays; their ability to interact with the binding proteins was expressed as relative competitive index (RCI) values where the RCI of 1,25(OH)₂D₃ is defined to be 100. The results indicate that the 25-hydroxyl group is the most important hydroxyl for the interaction of 1,25(OH)₂D₃ with this antiserum. The absence of this hydroxyl group decreases the RCI value to 0.2. Lack of the hydroxyl at carbon-3 or carbon-1 decreases the RCI value to 33 or 25, respectively, indicating that the specificity of this antiserum for the A ring is much lower than for the side chain. The high specificity for the side chain is underlined by the fact that insertion of an additional hydroxyl group at C-24 or C-26 of 1,25(OH)₂D₃ decreases the binding affinity to the antiserum markedly. The chick intestinal mucosal receptor shows a comparable high specificity for the side chain of 1,25(OH)₂D₃, but an even higher specificity for the A ring in comparison to the antiserum. With the intestinal receptor, the 3-hydroxyl is only 1/ 10th as important as the 1-hydroxyl group and the 25-hydroxyl group for the binding process. Scatchard analysis showed a K_D value of 1.7 × 10^?10m for the antiserum and 2.3 × 10^?10m for the chick intestinal mucosal receptor for the equilibrium binding of 1,25(OH)₂D₃ at 2 °C. The association rate constant at 2 °C was determined to be 5.8 × 10⁷ M^?1 min^?1 for the antiserum and 0.55 × 10⁷ M^?1 min^?1 for the receptor, indicating a 10-fold more rapid association of 1,25(OH)₂D₃ to the antiserum in comparison to the receptor. Furthermore, the dissociation process was found to be slower for the chick intestinal receptor (dissociation rate constant 3.6 × 10^?5 min^?1 versus 21.0 × 10^?5 min^?1).     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

The chick intestinal cytosol binding protein for 1,25-dihydroxyvitamin D3: A study of analog binding

The structural features of 1,25-dihydroxyvitamin D₃ that permit its high affinity binding to a 3.7 S protein from chick intestinal cytosol were determined in a series of binding and competition experiments analyzed by sucrose density gradient centrifugation. Optimal binding to the 3.7 S protein was achieved when both 1α- and 25-hydroxyls were present in the vitamin D₃ molecule. Modification of the side chain by the introduction of a methyl on C-24 and a double bond on C-22,23 (1,25-dihydroxyvitamin D₂) did not alter the binding of 1,25-dihydroxyvitamin D₃, but significantly diminished the binding of 25-hydroxyvitamin D₃. However, introduction of a hydroxyl on C-24 decreased the ability of either 1,25-dihydroxyvitamin D₃ or 25-hydroxyvitamin D₃ to compete, especially when the 24-hydroxyl was in the S configuration. These results reveal that the 3.7 S protein requires specific ligand structural features for binding and suggest that metabolite discrimination by the chick intestinal receptor system is likely located in the 3.7 S cytosol protein.     

In vitro inhibitor studies of vitamin D 25-hydroxylase in rat liver microsomes

The ability of four vitamin D analogs to inhibit the liver microsomal vitamin D-25-hydroxylase was determined. 19-Hydroxy-10(S),19-dihydrovitamin D3,25-fluorovitamin D3, 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide and 25-aza-vitamin D3 were competitive inhibitors with apparent KI values of 44, 137, and 870 nM, and 6.4 microM, respectively. The values for the 19-hydroxy-10(S), 19-dihydrovitamin D3, 25-fluorovitamin D3, and 25-aza-vitamin D3 correspond well to other literature reports with respect to their relative in vivo inhibitory properties. 24-Oxovitamin D3 oxime also proved to be a potent inhibitor but a detailed analysis was prohibited by the lack of material. The 3 beta-hydroxy-9,10-seco-5,7,10(19)-choletrien-24-oic acid dimethylamide was also tested in vivo but had no antagonistic activity when provided at a 2000-fold excess over vitamin D3.     

The biological activity of rat intestinal retinoic acid metabolites in the vaginal smear assay

Vitamin A-deficient rats were given a single intrajugular injection of 1 mg all-trans-[11-³H]retinoic acid and 3 h later the rats were killed. The small intestines were extracted and chromatographed by high-performance liquid chromatography to yield distinct metabolites. These were quantitated using the assumption that the specific activity of the metabolite is equal to that of the parent [³H]retinoic acid. The biological activity of all discernible metabolities was determined in the vitamin A-deficient female rat by vaginal smear assay. Retinoic acid and retinoyl-β-glucuronide from the preparation had equal activity while no activity was found for any of the other metabolite fractions. Thus, no evidence for an unknown metabolite having potent epithelial differentiating activity could be found in this target tissue of vitamin A action.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D₃, 24,24-difluoro-25-hydroxyvitamin D₃ has been synthesized and shown to be equally as active as 25-hydroxyvitamin D₃ in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D₃ was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D₃, while large amounts were found when 25-hydroxyvitamin D₃ was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D₃, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D₃ from 24,24-difluoro-25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D₃ is less active than 25-hydroxyvitamin D₃ and 24R,25-dihydroxyvitamin D₃. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D₃ is more active than 25-hydroxyvitamin D₃ which is itself more active than 24R,25-dihydroxyvitamin D₃. The 24,24-difluoro-1,25-dihydroxyvitamin D₃ is equal to 1,25-dihydroxyvitamin D₃, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D₃ in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D₃ cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.     

An in vitro study of the stability of the chicken intestinal cytosol 1,25-dihydroxyvitamin D3-specific receptor

The stability of the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D₃ has been examined using radiological binding studies and sucrose density gradient ultracentrifugation. Specific binding of 1,25-dihydroxyvitamin D₃ to the 3.7 S binding protein decreases in crude cytosol in a time- and temperature-dependent manner. Increased receptor instability is also observed outside a pH range of 6 to 10. Ionic strength does not seem to be a critical factor in preventing loss of specific 1,25-dihydroxyvitamin D₃ binding activity. However, when KCl is present at a concentration of 300 mm during cytosol preparation, quantitatively more specific binding per unit protein was obtained. Consistent with the idea that loss of specific binding might be due to enzymatic degradation or inactivation of receptor, dilution of cytosol was found to slow the rate of loss of specific 1,25-dihydroxyvitamin D₃ binding. The importance of maintaining a reducing environment for the 1,25-dihydroxyvitamin D₃ binding protein is demonstrated by the destruction of binding activity by n-ethylmaleimide and by the increased stability in the presence of 5.0 mm dithiothreitol. Likewise, 5.0 mm monothioglycerol was partially effective in preventing the loss of specific 1,25-dihydroxyvitamin D₃ binding during in vitro incubation. Several protease inhibitors were not able to exert a stabilizing influence on receptor integrity during in vitro incubations. Albeit, both tosylamide-phenylethylchloromethyl ketone and p-hydroxymercuribenzoate actually decreased specific 1,25-dihydroxyvitamin D₃ binding. This inhibition appeared to be reversible if samples were subsequently incubated in the presence of dithiothreitol. These results clearly demonstrate that the aporeceptor is extremely unstable and the integrity of sulfhydryl constituents is of primary importance.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

Characterization of 1,25-dihydroxyvitamin D3-receptor complex interactions with DNA by a competitive assay

To identify and assess the specificity of the 1,25-dihydroxyvitamin D₃ chick intestinal cytoplasmic receptor's nucleotide binding site, a competitive DNA-cellulose binding assay was utilized. Unlike other steroid hormone receptors, the 1,25-dihydroxyvitamin D₃-receptor complex binds homologous DNA at 4 °C and does not appear to undergo thermal- or salt-induced activation. Data are presented which suggest that receptor binding discriminates between double-stranded DNA and RNA but is not specific with respect to DNA base sequences. However, DNA base sequence selectivity by 1,25-dihydroxyvitamin D₃-receptor complexes is observed using synthetic polydeoxyribonucleotides, particularly, poly(dA-dT) · poly(dA-dT) and poly(dA) · poly(dT). Preference for double-stranded over single-stranded DNA was also observed. Consistent with this finding, both actinomycin D and ethidium bromide caused a dose-dependent inhibition of receptor binding to DNA-cellulose. It is concluded that the 1,25-dihydroxyvitamin D₃-receptor complex has specificity for AT-rich segments of double-stranded DNA and that this interaction is not merely electrostatic, but also involves hydrophobic interaction with the major and/or minor grooves of the DNA helix.     

Crystallization of hen eggwhite riboflavin-binding protein

Crystals of hen eggwhite riboflavin-binding protein have been grown by equilibrium dialysis in solutions buffered with 0.05 m-Tris · HCl (pH 8.5) using ammonium sulphate as the precipitant. The crystals belong to the space group P3₁21 (or enantiomorph) with $\text{a = b = 112.5}\text{A}\text{?}\text{and}\text{c = 72.0}\text{A}\text{?}\text{,}$ and diffract to a resolution of 2.8 Å.     

N-acetyl conjugates of basic amino acids newly identified in rat urine

Ninhydrin-negative conjugates of basic amino acids were isolated from rat urine and were characterized. The following conjugates of basic amino acids are the compounds newly identified in animal urine specimens, N^α-acetyl-N^π-methylhistidine, N^α-(N-acetyl-β-alanyl)histidine (N-acetylcarnosine), N^α-acetyl-N^G,N^′G-dimethylarginine, N^α-acetyl-N^G,N^G-dimethylarginine, and N^α-acetyl-N^?,N^?,N^?-trimethyllysine.     

The structure of a platinum(II) complex of cytidine-3'-monophosphate.

The structure of the cis-[Pt(NH₃)₂(3′-CMP)₂]^2? ion, isolated in a partially protonated form as its cesium salt, has been analyzed by single-crystal x-ray diffraction methods. The 3′-CMP ligands bind in a monodentate fashion through their N(3) atoms: in contrast to the structure of [Pt(en)(5′-CMP)]₂, no covalent platinum-phosphate bonding is found. This compound represents the first example of a 1:2 cis-metal/cytosine complex structurally characterized.     

24,24-Difluoro-1,25-dihydroxyvitamin D3: In vitro production,isolation, and biological activity

24,24-Difluoro-1,25-dihydroxyvitamin D₃ has been synthesized by in vitro incubation of vitamin D-deficient chick kidney homogenates with 24,24-difluoro-25-dihydroxyvitamin D₃. The compound produced was isolated and purified by successive high-performance liquid chromatographic steps and then identified by means of ultraviolet absorption spectrophotometry and mass spectrometry. The difluoro analog of 1,25-dihydroxyvitamin D₃ is found to be highly active in stimulating intestinal calcium transport and bone calcium mobilization in vitamin D₃-deficient rats.     

Control of the mitochondrial inner membrane permeability by sulfhydryl groups

The effect of some thiol alkylating agents (N-substituted maleimide derivatives) on the permeability of the mitochondrial inner membrane was investigated. Several experimental approaches were used to study the modifications of the permeability properties. Alkylation of sulfhydryl groups led to an increase in the nonspecific permeability as judged by (i) the augmentation of the rate of osmotic shrinkage of mitochondria induced by polyethylene glycol, (ii) the sensitization of succinate dehydrogenase toward oxaloacetate, (iii) the enhancement of the oxidation rate of exogenous NADH, and (iv) the increase of the sucrose permeable space. The sulfhydryl groups involved in the maintenance of the selective permeability were shown to be located in the hydrophobic core of the membrane. Energization of mitochondria provoked an unmasking of these sulfhydryl groups. When magnesium ions were present in the incubation medium, N-substituted maleimide derivatives promoted gross modifications of the intramitochondrial ionic contents. Effluxes of endogenous calcium ions, inorganic phosphate, adenine nucleotides, and NAD(P)H were established. It was concluded that sulfhydryl groups probably play a crucial role in the maintenance of the membrane integrity and thus control the mitochondrial inner membrane permeability.     

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.