Metabolism of the anti-obesity agent, 4-amino-5-ethyl-3-thiophenecarboxylic acid methyl ester hydrochloride, in rats and dogs.

1. In 24 h, male rats excreted in urine 1% of an intra-gastric 100 mg/kg dose of 4-amino-5-ethyl-3-[4-14C]thiophenecarboxylic acid methyl ester hydrochloride (I) as unchanged I and 59% as 4-amino-5-ethyl-3-thiophenecarboxylic acid (II), mostly conjugated. 2. In rats dosed intra-duodenally with I (50 mg/kg), little I was found in the systemic circulation (less than 2 micrograms/ml) but high concentrations (26 micrograms/ml) were present at five minutes in portal plasma. At five minutes, II was found at 89 and 93 micrograms/ml in systemic and portal plasma, respectively. First-pass ester hydrolysis by the duodenum and liver may explain the near absence of I and the high concentrations of II in systemic plasma. 3. Dogs which received 30 mg/kg 14C-I intra-gastrically, excreted 0.3% I, 30.8% II and 6.8% as 5-ethyl-4-(methylamino)-3-thiophenecarboxylic acid (III), the N-methyl derivative of II. 4. Dogs which received approximately equivalent intra-venous or intra-gastric doses of non-radioactive I and II had high plasma concentrations of II but only small concentrations of I. Plasma concentrations of II after intra-gastric doses of non-radioactive I or II were similar, indicating that both compounds are pharmacokinetically equivalent. I may be a prodrug of II.

Use of Fourier transform 1H NMR in the identification of vitamin D2 metabolites.

The use of Fourier transform 1H NMR to characterize vitamin D2 metabolites is described. A 300-MHz spectrometer capable of generating a 6-microseconds pulse and a sweep width of 4000 Hz was used. High-resolution spectra were obtained on 5 micrograms of material using standard 5-mm NMR tubes fitted with glass inserts and isotopically enriched chloroform-d solvent. The data acquisition time under these conditions was 4 h. Application of this technique to a variety of both synthetic and naturally occurring vitamin D2 metabolites, in addition to synthetic delta 22-1,25-dihydroxyvitamin D3, resulted in the reassignment of the chemical shifts for the C-21 and C-28 methyl groups of vitamin D2. The C-21 methyl group resonance is now assigned to the doublet appearing at delta 1.01, whereas the C-28 signal corresponds to the doublet at delta 0.90. An examination of the spectrum of 24 (R),25-dihydroxyvitamin D2 also led to the reassignment of the side-chain methyl group resonances. This technique is an additional means of identifying microgram quantities of vitamin D metabolites.

Stimulatory effect of 1 alpha, 25-dihydroxyvitamin D3 on the formation of skin tumors in mice treated chronically with 7,12-dimethylbenz[a]anthracene.

The effect of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) and its 24,24-difluoro analog on the formation of skin tumors in mice was evaluated in a complete carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as the carcinogen. Twice weekly topical application of 0.25-0.50 nmol of 1 alpha, 25-(OH)2D3 or 0.05-0.10 nmol of the difluoro analog of 1 alpha, 25-(OH)2D3 1 hour prior to treatment with 50 nmol DMBA stimulated tumor formation several fold compared to animals receiving DMBA alone. Topical application of 0.50 nmol of 1 alpha, 25-(OH)2D3 24 hours after treatment with DMBA, or half of this dose of the vitamin D3 metabolite, applied 1 hour before and 24 hours after treatment with DMBA, also stimulated tumor formation several fold. These results are in marked contrast to the potent inhibitory effect of 1 alpha, 25-(OH)2D3 and its difluoro analog on the formation of skin tumors in mice promoted by 12-O-tetradecanoylphorbol-13-acetate.

(23S)-1,23,25-Trihydroxyvitamin D3: its biologic activity and role in 1 alpha,25-dihydroxyvitamin D3 26,23-lactone biosynthesis.

(23S)-1,23,25-Trihydroxyvitamin D3 was isolated from bovine kidney homogenates incubated with 1,25-dihydroxyvitamin D3 by sequential chromatography through one Sephadex LH-20 column and three high-performance liquid chromatography systems. Ultraviolet absorption spectroscopy and mass spectrometry confirmed the structural assignment. One high-performance liquid chromatography system separated the R and S epimers of 1,23,25-trihydroxyvitamin D3 and indicated that the natural product had the S configuration. Plasma pharmacokinetic studies in rats showed that (23S)-1,23,25-trihydroxy[3H]vitamin D3 was rapidly cleared from plasma (t1/2 = 60 min). 1 alpha,25-Dihydroxy[3H]vitamin D3 26,23-lactone appeared concurrently with the disappearance of (23S)-1,23,25-trihydroxy[3H]vitamin D3. Experiments with radioinert compounds showed that 1,25-dihydroxyvitamin D3 and (23S)-1,23,25-trihydroxyvitamin D3 were efficient precursors to 1,25-dihydroxyvitamin D3 26,23-lactone both in intact and in nephrectomized rats. (25S)-1,25,26-Trihydroxyvitamin D3, however, was ineffective at raising plasma 1,25-dihydroxyvitamin D3 26,23-lactone concentrations. These results confirm the presence of extrarenal 1,25-dihydroxyvitamin D3 23(S)-hydroxylase(s) and demonstrate that C-23 hydroxylation of 1,25-dihydroxyvitamin D3 precedes C-26 hydroxylation in the formation of 1,25-dihydroxyvitamin D3 26,23-lactone. (23S)-1,23,25-Trihydroxyvitamin D3 had no intestinal calcium absorptive or bone calcium resorptive activity when dosed to vitamin D deficient rats at levels up to 500 ng. C-23 oxidation, therefore, appears to be a physiologic pathway of 1,25-(OH)2D3 metabolism and a major pathway for the deactivation of pharmacologic levels of 1,25-dihydroxyvitamin D3.

A simple method for generation of antibodies with specificity for 1,25-dihydroxyergocalciferol and 1,25-dihydroxycholecalciferol.

A simple method for production of antisera with high affinity and selectivity for 1 alpha, 25-dihydroxyergocalciferol and 1 alpha, 25-dihydroxychole-calciferol is described. 1 alpha-Hydroxy-25,26,27-trisnorcholecalciferol-24-oic acid was coupled directly to bovine serum albumin. Rabbits immunized with this conjugate rapidly produced antibodies that bound 3H-1 alpha,-25-dihydroxycholecalciferol with high affinity and demonstrated nearly equal reactivity with 1 alpha, 25-dihydroxyergocalciferol and poor reactivity with 25-hydroxycholecalciferol; 24,25-dihydroxycholecalciferol; 25,26-dihydroxycholecalciferol; and 1 beta,25-dihydroxycholecalciferol. The use of one of these antisera has led to the development of a specific assay for 1 alpha,25-dihydroxyergocalciferol and 1 alpha,25-dihydroxycholecalciferol in human serum.

A radioimmunoassay for 1,25-dihydroxycholecalciferol.

We describe a radioimmunoassay for 1,25-dihydroxycholecalciferol in human serum. We raised antisera in rabbits to 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine serum albumin, and obtained sensitive, high-titer antibodies. These antibodies had a high affinity for 1,25-dihydroxycholecalciferol and cross reacted mainly with 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. Addition of 1 mL of normal rabbit serum per liter reduced this interference to 5 and 4%, respectively. However, these interfering steroids are present in large excess, so extensive purification of 1,25-dihydroxycholecalciferol from serum is necessary. The steroid was extracted with ethyl acetate/cyclohexane, purified on Sephadex LH-20, and then chromatographed on a column of silicic acid. The radioimmunoassay is sensitive to 5 pg/tube (3 ng/L of serum). The between-assay CV was 14%. The mean concentration of 1,25-dihydroxycholecalciferol in the serum of 54 healthy adults was 38 (SD 12) ng/L, with no sex-related difference. The assay was further validated by the finding of low or undetectable concentrations in patients with chronic renal failure and of increased concentrations in the serum of patients with primary hyperparathyroidism. In comparison with previously described methods, the major advantage of the present assay is the use of stable gamma-globulins, which are available in large amounts, as binding protein.

A radioimmunoassay for 1,25-dihydroxycholecalciferol.

1. 1alpha,25-Dihydroxy-25-hemisuccinate cholecalciferol has been synthesized and conjugated to bovine serum albumin. 2. This conjugate is immunogenic; when injected into rabbits antibodies of high affinity for 1alpha,25-dihydroxycholecalciferol were obtained. 3. Vitamin D metabolites lacking the 1alpha-hydroxy group were of lower cross-reactivity with the antibodies. 4. By using these antibodies and 1alpha,25-[23,24-3H]dihydroxycholecalciferol as tracer a sensitive radioimmunoassay has been developed capable of detecting 20 pg of 1alpha,25-dihydroxycholecalciferol.