Human pharmacokinetic profile of 1,25-dihydroxyvitamin D3-glycoside of herbal origin.

A natural form of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, was identified in glycosylated form in Solanum glaucophyllum (SG). Solbone P, an extract of SG with high and homogenous content of glycosylated 1,25(OH)2D3, was chemically characterized and produced under GMP conditions. Three different doses of glycosylated 1,25(OH)2D3 were given as single oral dose to 16 healthy volunteers in a first-in-man trial. The oral pharmacokinetic properties of 1,25(OH)2D3 of SG origin were established and the subjects were monitored until day 28 for safety reasons. This included regular monitoring of vital signs, electrocardiogram (ECG) data, calcium, phosphate and creatinine values. Subjects were exposed to up to the equivalent of a 40-fold level of the recommended human daily dose for synthetic 1,25(OH)2D3 (0.5µg/subject/day) without experiencing any untoward effects. When compared with the historically established pharmacokinetics profile of synthetic 1,25(OH)2D3, glycosylated 1,25(OH)2D3 of herbal origin exhibited delayed absorption characteristics. The phenomenon is species independent, as similar pharmacokinetic patterns were observed in rats and chickens. This modified release pattern may be attributed to the glycosylation of herbal 1,25(OH)2D3 because de-glycosylation by ubiquitous intestinal enzymes prior to intestinal uptake of the unmodified 1,25(OH)2D3 is the rate-limiting step. The major relevance of this finding is that the human pharmacokinetic profile of glycosylated 1,25(OH)2D3 of herbal origin is reminiscent of a delayed release formulation of free 1,25(OH)2D3, resulting in a wider therapeutic window, a potentially longer therapeutic effectiveness, and thus, a better pharmacologic tolerance. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

A new angiotensin-II- receptor blocker, CGP 48933: local tolerance and effect on intraocular pressure. A pilot study.

CGP 48933, a new angiotensin-II-receptor antagonist, has been shown to lower intraocular pressure (IOP) in two different rabbit glaucoma models in a dose-dependent manner after local application. As a further step a pilot study was performed in human eyes. The trial consisted of three parts. Parts 1 and 2 comprised a double-masked intraindividual trial between CGP 48933 and its vehicle (saline) in five healthy volunteers (Part 1) and five patients with early stages of primary open-angle glaucoma (Part 2), to assess local tolerance and the effect on IOP. Part 3 was a single-masked intraindividual trial between CGP 48933 and saline, to find the effective dose range of the new compound. Local tolerance was assessed as excellent in all subjects. No conjunctival hyperemia burning or itching occurred. There were no significant changes in IOP from baseline in drug or vehicle-treated eyes. In addition, there was no dose-dependent (200 micrograms to 1 mg) effect of CGP 48933 on IOP. Systemic blood pressure, heart rate and pupil size did not change during the observation period. Topical application of CGP 48933 in its present formulation is thus not suitable for lowering IOP in humans.

The renin-angiotensin system in the rabbit eye.

We analyzed the components of the renin-angiotensin system (RAS) in ocular tissues of normal rabbit eyes and compared the results with those measured in rabbit eyes with proliferative vitreoretinopathy and ocular hypertension. Proliferative vitreoretinopathy was induced by injection of human platelets into the vitreous humor, and ocular hypertension was induced by injection of alpha-chymotrypsin into the posterior chamber. Angiotensinogen, renin, angiotensin converting enzyme (ACE), angiotensin II (Ang II), and Ang II receptors were assessed using conventional biochemical techniques. The vascularized tissues of normal eyes contained high renin and ACE activities concomitant with low concentration of angiotensinogen and Ang II. In general, in the ocular humors, the opposite was found. The Ang II receptor density was highest in the uveal tract [range 35-190 fmol/mg protein]. The AT1 receptor subtype predominated [> 80%]. The RAS was only minimally different in the two pathological models except that, in ocular hypertension, the renin activity in the uveal tract was reduced [-50%]. Also, the ratio of AT1 to AT2 receptors changed as compared to control, although the total receptor density remained unaltered. In conclusion, we present evidence for the presence of a complete local RAS in the rabbit eye, which is only marginally affected by the two pathological models studied.

Kinetics of hydroxyethyl starch in horses.

In a controlled study, the distribution and elimination kinetics of hydroxyethyl starch as well as clinically relevant parameters were determined in horses. The half-life of the first phase was 5.59 hours, that of the second phase 122.22 hours. During the first phase, hydroxyethyl starch persisted almost exclusively intravascularly. The results of this study are largely in agreement with those in human beings. Thus, routes of elimination, duration of plasma-expanding action, distribution volume and redistribution kinetics in horses and human beings are very similar. However, the elimination kinetics of the second phase and the behavior of serum amylase appear to be equine-specific. Coagulation is barley influenced by the administration of hydroxyethyl starch. The results of this study confirm that hydroxyethyl starch is very suitable for use as a plasma-expander in horses.

Kinetics of uptake and effects of topical indomethacin application on protein concentration in the aqueous humor of dogs.

The pharmacokinetic properties of indomethacin and its effects on aqueous protein values were studied in 15 clinically normal Beagles. The dogs were treated every 6 hours with 1% indomethacin suspension in 1 eye, with the other eye serving as a control. After 24 hours, the dogs were anesthetized and samples of aqueous humor (AH) were drawn by aqueocentesis at 0, 15, 30, 60, and 90 minutes after initial paracentesis. Additional samples were drawn at the time of euthanasia, 180 (6 dogs) and 360 minutes (9 dogs) minutes after initial paracentesis. Blood samples were obtained at each treatment and at each aqueocentesis. The eyes were enucleated after dogs were euthanatized. Aqueous protein concentrations and indomethacin concentrations in AH, plasma, and different ocular tissues were determined. Topical indomethacin administration had no effect on baseline protein concentrations of AH. It reduced protein concentrations in AH significantly at all times after initial aqueocentesis. This reduction was approximately 30%. Indomethacin in the AH is mostly protein-bound. Concentrations were 350 ng/ml in primary AH and 1,305 ng/ml in secondary AH, 90 minutes after initial aqueocentesis. Free-drug concentrations were relatively constant at about 220 ng/ml. Indomethacin administered topically is readily absorbed by the ocular adnexae, reaching a steady-state concentration of 25 ng/ml in blood plasma 18 hours after the start of treatment. Plasma concentrations were 50 times lower than therapeutically effective concentrations. High indomethacin concentrations were found in the cornea only. Low concentrations were found in the iris and ciliary body, the lens, and in the choroid.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of multiple proteins structurally related to gamma-glutamyl transpeptidase in non-neoplastic adult rat hepatocytes in vivo and in culture.

Freshly isolated hepatocytes from normal adult rat liver do not express measurable gamma-glutamyl transpeptidase (GGT) mRNA in contrast to the significant GGT mRNA levels expressed by normal adult rat kidney and hyperplastic bile ductular tissue from bile duct-ligated rats. However, the induction of GGT activity in rat hepatocytes by two-thirds hepatectomy was accompanied by the appearance of a high level of GGT mRNA. We are now able to demonstrate that normal adult rat hepatocytes express 5 protein bands which cross-react with 2 different anti-rat kidney GGT antisera. The apparent molecular weights were 26.9, 58.0, 63.9, 73.5, and 83.4 kDa, respectively. Expression of the 26.9- and 58.0-kDa proteins strikingly parallels the pattern of induction of GGT enzymatic activity. This suggests that these 2 proteins correspond to the active dimeric enzyme previously described in kidney and neoplastic hepatocellular tissue. In normal hepatocytes, the 73.5-kDa protein represents 50% of the total GGT-immunoreactive protein, in contrast to kidney, where this band contains less than 4% of the GGT protein. The kinetics of expression of the 73.5-kDa protein upon induction of GGT activity in hepatocytes, as well as in culture turnover studies, suggests that this protein is a precursor form of the active enzyme, such as the described 78/79-kDa single-chain glycoprotein propeptide of GGT. It appears that in normal hepatocytes, this precursor is not processed to the same extent as in kidney or in hyperplastic bile ductular tissue.

[Pharmacologic effects of biotin on epidermal cells]. / Pharmakologische Wirkungen von Biotin auf Epidermiszellen.

Biotin deficiency in animals causes pathological changes of the skin and its appendages including, for example, exfoliative dermatitis, depigmentation, and alopecia. The hooves of biotin-deficient swine are weak, brittle, and often necrotic. These changes disappear after dietary biotin supplementation. Biotin supplementation also noticeably improves the hoof quality of horses, cattle and swine having no apparent biotin deficiency. In order to elucidate the molecular basis of these effects, the influence of biotin on cytokeratin expression in a keratinocyte cell line (Ha-CaT) was investigated using electrophoretic and immunological techniques. Pharmacological biotin concentrations of 1 microM, and 100 microM in the culture medium caused a specific increase in cytokeratins, which are normally induced upon terminal differentiation of epidermal cells in vivo. The expression of cytokeratins occurring in stratified epithelia independent of differentiation were not affected. These findings show that biotin directly stimulates the differentiation of epidermal cells. Such a molecular mechanism revealed in cell culture could provide an explanation for the therapeutic effects of pharmacological doses of biotin on hoof quality in farm animals.

17 Beta-hydroxysteroid UDP-glucuronosyl transferase is expressed in bile ductular epithelial cells under physiological conditions.

Using an improved procedure to isolate pure bile ductular epithelial cells from rat liver, we were able to define UDP-glucuronosyl transferase (UDPGT) isozymes expressed in these cells under physiological conditions. The cells contained mRNA for a 17 beta-hydroxysteroid, a 3 alpha-hydroxysteroid and a phenol UDPGT. Concomitantly, a distinct pattern of UDPGT-immunoreactive proteins was expressed, including a putative 17 beta-hydroxysteroid UDPGT. The presence of this UDPGT isozyme was confirmed by a high level of testosterone glucuronidation activity. This is the first demonstration of a metabolic pathway in bile ductular epithelial cells that is primarily dedicated to the processing of endogenous compounds.

Effects of medium and substratum conditions on the rates of DNA synthesis in primary cultures of bile ductular epithelial cells.

Select medium and substratum conditions were investigated for their effects on semiconservative DNA synthesis in essentially pure primary cultures of bile ductular epithelial cells that were initially isolated from cholestatic rat livers at 6 to 10 wk after bile duct ligation. DNA synthesis in these cultured cells was serum-dependent, being at its highest level when the concentration of fetal bovine serum present in the medium was maintained at 10%. This serum-dependent DNA synthesis was completely inhibited when 10 mM hydroxyurea was also included in the medium, and bile ductular cells cultured in the continued presence of 1.0% fetal bovine serum showed only marginal DNA synthesis during 8 to 10 d of primary culture when compared with no-serum controls. Maximum rates of serum-dependent DNA synthesis were obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with either fibronectin from bovine plasma or type I rat-tail collagen. Cells cultured on plastic coated with basement membrane Matrigel exhibited the lowest levels of DNA synthesis, whereas those on plastic alone had intermediate amounts. Furthermore, the addition of epidermal growth factor (50 ng.ml-1.d-1) to medium supplemented with 1.0% fetal bovine serum greatly enhanced the rate of DNA synthesis in bile ductular cells after 6 d in primary culture on type I collagen-coated plastic over that measured in solvent control cultures. These findings indicate that our bile ductular epithelial cell culture model is potentially useful in the study of biliary cell growth regulation and carcinogenesis.

Isolation of 8-methoxypsoralen accessible DNA domains from chromatin of intact cells.

The chromatin organization of living mammalian cells was probed using 8-methoxypsoralen (MOP). In intact cells, MOP intercalates into DNA domains which are also preferentially accessible to micrococcal nuclease. After UV365 nm irradiation of MOP-treated cells, this chemical forms bifunctional adducts crosslinking the two strands of DNA. Following extraction of cellular DNA, heat denaturation and renaturation at low temperature, the fraction of crosslinked DNA is obtained following enzymatic hydrolysis of unhybridized, non-crosslinked DNA by nuclease S1 treatment. An application of this procedure in the isolation of 8-methoxypsoralen-accessible DNA domains during DNA excision repair is shown.

Isolation, culture, and transplantation of intrahepatic biliary epithelial cells and oval cells.

Recent advances made in the isolation, culture, and transplantation of defined populations of intrahepatic biliary epithelial cells and oval cells have permitted direct analysis of the functions, growth properties, and differentiation potential of these respective cell types in their untransformed or transformed states. This review provides a current and comprehensive examination of the various approaches that have been taken to isolate and culture intrahepatic biliary epithelial cells from normal and cholestatic liver and oval cells from preneoplastic liver. Emphasis is placed on comparing the phenotypic features and growth properties of these various biliary cell types in vitro as well as on describing their transplantation into ectopic tissue sites. In addition, the oval cell is evaluated in terms of its potential role as a 'facultative stem cell' during hepatocarcinogenesis.

[Intravenous congestion anesthesia/-antibiotic administration in cattle--indications,technics, complications]. / Die intravenöse Stauungsanästhesie/-antibiose beim Rind--Indikationen, Technik, Komplikationen.

We present a literature review on current techniques of intravenous regional anesthesia and intravenous regional antibiosis of the distal limb in cattle. Our own experiences performing a combined procedure of intravenous anesthesia and antibiosis (10 million I.U. benzylpenicillin sodium dissolved in 15-20 ml 2%-lidocaine hydrochloride) are discussed in detail. Complete anesthesia of the treated limb was achieved in 22 out of 23 cases (96%). The successfully treated animals did not express any symptoms of pain for the entire surgical procedure. In 2 out of 15 patients (13%) we observed serious post-surgical complications. The reason of which was extensive thrombosis of all veins distal of the tourniquet. The age of the clots at the time of slaughtering of the cows was determined histologically. A direct cause effect relationship between intravenous anesthesia/antibiosis and complication is indicated. We conclude that direct toxicity of the 2000-fold overdose of benzylpenicillin (as compared to generally accepted therapeutic levels) most likely caused the problem. We recommend to reduce the dose of penicillin in regional intravenous antibiosis to maximally 100,000 I.U., even in the case of local sepsis.

Enzyme profile of rat bile ductular epithelial cells in reference to the resistance phenotype in hepatocarcinogenesis.

An extensive bile ductular cell hyperplasia with the formation of well-differentiated bile ductules is the most prominent feature of rat liver at 6 to 15 weeks after bile duct ligation. We have improved our previous cell isolation procedure and are now routinely able to obtain from such livers high yields of viable bile ductular epithelial cells. These cells were characterized with respect to their specific activities of gamma-glutamyl transpeptidase and beta-glucuronidase and of select Phase I and Phase II enzymes of biotransformation. At the time of their isolation, only a very small number of the bile ductular epithelial cells were observed to be in DNA synthesis. In addition, in histological sections prepared from intact hyperplastic bile ductular tissue isolates, only the bile ductular epithelial cells exhibited histochemical staining for gamma-glutamyl transpeptidase activity. Typically, greater than 95% of the cells isolated from this tissue were also found to be histochemically positive for gamma-glutamyl transpeptidase activity, and no hepatocytes were seen contaminating this cell population. Biochemically, the isolated bile ductular cells exhibited a gamma-glutamyl transpeptidase specific activity that was 100 times higher than that of hepatocytes isolated at the same time from the bile duct-ligated rats and more than 300 times higher than the specific activity of the enzyme of freshly isolated normal rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characteristics of hyperplastic rat bile ductular epithelial cells cultured "on top" and "inside" different extracellular matrix substitutes.

An essentially pure population of bile ductular epithelial cells isolated from bile duct-ligated rats was placed in primary culture by plating the cells either "on top" of or "inside" different extracellular matrix substitutes, including basement membrane Matrigel, type I collagen gel, and agarose gel. Plating efficiencies of greater than 60% were obtained when the cells were seeded in the presence of 1.0% fetal calf serum on top of Matrigel and collagen gel, but there was very little if any cell attachment to the agarose surface. In contrast, the cells could be maintained equally well and at very similar densities when they were cultured inside the various gel substances, including agarose. Regardless of substratum condition, bile ductular cells at 10 days in primary culture expressed specific activities of the marker enzymes gamma-glutamyl transpeptidase and leucine aminopeptidase which were significantly higher than those shown by freshly isolated cells. On the other hand, alkaline phosphatase activity of the cells became undetectable by day 3 of culture when they were cultured on top of either Matrigel or collagen gel but was retained at approximately 50% of its original level in cells cultured for 10 days within Matrigel or agarose gel. Treatments with dexamethasone or hydrocortisone (i.e., 10(-6) M) inhibited the increase in gamma-glutamyl transpeptidase activity of the cultured cells but did not affect the other enzyme changes. Subcultures of the bile ductular epithelial cells were developed by passing the cells in the presence of 10% fetal calf serum on surfaces coated with either type I collagen or Matrigel. In either case, cells subjected to at least 4-6 passages (up to 100 days of culture) were still characterized by a high gamma-glutamyl transpeptidase activity. Preliminary results obtained with cells plated at very low density within Matrigel also indicated the development of cell growths that appeared to be organized in the form of distinct acinar-like structures.