Comprehensive taxonomy and worldwide trends in pharmaceutical policies in relation to country income status.

BACKGROUND: Rapidly evolving socioeconomic and technological trends make it challenging to improve access, effectiveness and efficiency in the use of pharmaceuticals. This paper identifies and systematically classifies the prevailing pharmaceutical policies worldwide in relation to a country's income status. METHODS: A literature search was undertaken to identify and taxonomize prevailing policies worldwide. Countries that apply those policies and those that do not were then grouped by income status. RESULTS: Pharmaceutical policies are linked to a country's socioeconomics. Developed countries have universal coverage and control pharmaceuticals with external and internal price referencing systems, and indirect price-cost controls; they carry out health technology assessments and demand utilization controls. Price-volume and risk-sharing agreements are also evolving. Developing countries are underperforming in terms of coverage and they rely mostly on restrictive state controls to regulate prices and expenditure. CONCLUSIONS: There are significant disparities worldwide in the access to pharmaceuticals, their use, and the reimbursement of costs. The challenge in high-income countries is to maintain access to care whilst dealing with trends in technology and aging. Essential drugs should be available to all; however, many low- and middle-income countries still provide most of their population with only poor access to medicines. As economies grow, there should be greater investment in pharmaceutical care, looking to the policies of high-income countries to increase efficiency. Pharmaceutical companies could also develop special access schemes with low prices to facilitate coverage in low-income countries.

Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones.

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by 1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (alpha 1-->6)-fucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc beta 1-->2, Gal beta 1-->4GlcNAc beta 1-->2 or Gal alpha 1-->3G alpha 1 beta 1-->4GlcNAc beta 1-->2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1-->2)-linked GlcNAc residues. Galactosylation of the GlcNAc beta 1-->2Man alpha 1-->6 branch occurs four times more frequently than that of the GlcNAc beta 1-->2Man alpha 1-->3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), exclusively (alpha 2-->6)-linked to beta Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two chimeric antibodies, but that the relative amounts of the major oligosaccharide components, the degree of sialylation and the molar ratio of Neu5Ac to Neu5Gc varies with the SP2/0 cell subclone, and only slightly with cell age.

Development of optimized transfectoma cell lines for production of chimeric antibodies in hollow fiber cell culture systems.

Methods for the selection of transfectoma cells that express large quantities of mouse-human chimeric antibodies have been develped. SP2/0 mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt)and the neomycin (neo) selection marker genes. ELISA-based screening of transfectoma clones resulted in the isolation of IgG-producing transfectomas. Introduction of the kappa light-chain 3'-enhancer into the light-chain expression vector significantly increased immunoglobulin expression, but only when the enhancer was located at its physiological site, 9 kb downstream of the kappa constant region exon. With some of the transfectomas, final yields of up to 80 mg/L of chimeric IgG were obtained in conventional flask cultures using serum-free growth medium. A pilot-scale AcuSyst Maximizer hollow fiber cell culture system was used for the production of gram amounts of chimeric IgG. Results obtained with different transfectoma clones in conventional culture were not fully predictive for yields in the hollow fiber system. In contrast, differences in productivity between individual clones in the laboratory-scale Tecnomouse cell culture unit were comparable with those in the Maximizer system. Up to 200 mg of chimeric IgG were produced per day in one Maximizer bioreactor. (c) 1995 John Wiley & Sons, Inc.

Oxytocin production and oxytocin messenger ribonucleic acid levels in bovine granulosa cells are regulated by insulin and insulin-like growth factor-I: dependence on developmental status of the ovarian follicle.

Oxytocin is a major peptide product of the ruminant corpus luteum, and the release of oxytocin from serum-free cultures of bovine granulosa cells is stimulated by insulin and insulin-like growth factor-I (IGF-I). Here we have assessed the effects of insulin and IGF-I on oxytocin gene expression in bovine granulosa cells and the dependence of these effects on the developmental status of the cells. When cells from individual follicles were cultured, the estradiol concentration of the follicular fluid was highly correlated with insulin-stimulated oxytocin release. Subsequently, cells were pooled from follicles selected on the basis of estradiol content, and two subsets of cells were distinguished. The first contained highly differentiated cells, as judged by the high estradiol (HE-cells) concentration of the follicular fluid (greater than 40 ng/ml), high levels of LH receptors, and high hCG-stimulated cAMP accumulation. The second subset contained cells from follicles with low estradiol (less than 1 ng/ml; LE-cells) which have fewer LH receptors and low hCG-stimulated cAMP accumulation. Oxytocin production was increased more than 50-fold by insulin (EC50, 230 +/- 57 ng/ml) and IGF-I (EC50, greater than 10 ng/ml), but only in the HE-cells. Oxytocin mRNA was also greatly increased by insulin and IGF-I in the HE-cells only. In contrast, insulin and IGF-I stimulated progesterone release from both HE- and LE-cells. Since oxytocin production is a characteristic of bovine luteal cells, our results support possible roles for IGF-I and insulin in regulation of luteinization or luteal activity. The data suggest that effects of insulin and IGF-I on oxytocin production reflect their effects on oxytocin gene transcription, and that granulosa cells must be appropriately primed (presumably by the in vivo hormonal environment) before they are able to produce oxytocin in response to these polypeptides.

Oxytocin and progesterone release from bovine corpus luteal cells in culture: effects of insulin-like growth factor I, insulin, and prostaglandins.

The ruminant corpus luteum synthesizes and secretes oxytocin, but little is known of the regulation of these processes in the ovary. In the present work we describe a method for the preparation of cells from the early bovine corpus luteum (1-5 days postovulation) and their maintenance in serum-free culture. The release of oxytocin and progesterone from these cells was increased by the addition of insulin or insulin-like growth factor I (IGF-I), but not by IGF-II. Hormone release (measured between 60 and 84 h of culture) was increased approximately 5-fold (oxytocin) and 2.5-fold (progesterone) by maximally effective concentrations of IGF-I (EC50, 0.27 nM) and insulin (EC50, 1.94 nM). Sustained exposure (0-84 h) to prostaglandins (PGs) caused a dose-dependent reduction in oxytocin release in the presence of IGF-I (PGF2 alpha EC50, 31 nM; rank order of potency, PGF2 alpha greater than PGE2 greater than PGE1), but did not markedly reduce progesterone release. The inhibitory effect of PG on oxytocin production was mimicked by sustained exposure to a protein kinase-C activator (phorbol 12,13-dibutyrate), supporting the proposed role for this enzyme as a mediator of PG action. These data provide the first demonstration that oxytocin release from early bovine corpus luteal cell cultures can be regulated by insulin, IGF-I, and PGs. Since granulosa and/or luteal cells produce and respond to IGF-I and PGF2 alpha, our data indicate functional interaction of these compounds in the regulation of luteal cell activity.

Hormonal modulation of pineal melatonin synthesis in rats and Syrian hamsters: effects of streptozotocin-induced diabetes and insulin injections.

Pineal levels of tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid and the enzyme activities of N-acetyltransferase and hydroxyindole-O-methyltransferase were determined in male albino rats and Syrian hamsters that were injected with insulin twice daily for three days, or injected with streptozotocin to induce diabetes. Neither insulin injections nor streptozotocin diabetes had any effect on pineal melatonin production in rats. In hamsters, diabetes reduced the nocturnal peak of pineal melatonin content by approximately one half, while insulin injections had no effect on pineal melatonin levels; however, insulin injections did cause a slight increase in pineal N-acetyltransferase activity. These findings indicate that the pineal gland of the hamster may be more sensitive to alterations in plasma insulin levels than the same organ in rats.

Diurnal changes in sensitivity to melatonin in intact and pinealectomized Djungarian hamsters: effects on thermogenesis, cold tolerance, and gonads.

Djungarian hamsters kept in long photoperiod (16:8 L:D) were injected daily at 0800, 1200, or 1600 with 25 micrograms of melatonin. During 90 days of treatment, body weight and fur coloration were checked at weekly intervals, and at the end of the treatment the reproductive status of the hamsters and their thermoregulatory properties (could limit, maximum thermoregulatory heat production, nonshivering thermogenesis, cytochrome oxidase activity in brown adipose tissue) were measured. Hamsters injected at 1600 changed from summer to winter status with regard to all functions investigated responding simultaneously; i.e., their body weights decreased, their fur became white, their gonads regressed, and their thermoregulatory properties improved. All these changes were identical to the effects of short photoperiod (8:16 L:D) exposure. Injections of melatonin at 0800 and 1200 were ineffective for reproductive functions, but the injection of melatonin at 0800 caused slight improvements of thermogenesis. The response to melatonin injected at 1600 could be suppressed by an additional injection of melatonin at 0800 (75 micrograms). Pinealectomized or ganglionectomized hamsters kept in long photoperiod did not respond to daily injections of melatonin at 1600 for the first 60 days of treatment, but during a prolonged treatment their sensitivity to melatonin was restored. Similarly, pinealectomized or ganglionectomized hamsters failed to respond to short photoperiod for about 40 days, but during prolonged exposure their sensitivity to short photoperiod was restored.

Effects of injections and/or chronic implants of melatonin and 5-methoxytryptamine on plasma thyroid hormones in male and female Syrian hamsters.

Plasma thyroid hormone concentrations in male and female Syrian hamsters were evaluated in five experiments after two pineal indoles, melatonin (MEL) and 5-methoxytryptamine (5-MT), were administered as chronic s.c. implants and/or daily afternoon injections. Circulating concentrations of thyroxine (T4), ordinarily maintained by the long photoperiod (LD 14:10), were inhibited by daily afternoon injections of 25 micrograms MEL but not 5-MT in male (experiment 1) and female (experiment 2) Syrian hamsters. The suppressive effect of MEL injections on T4 concentration in the long photoperiod was prevented in both sexes by a s.c. beeswax pellet containing either 1 mg MEL or 5-MT. In the third experiment, various doses of MEL or 5-MT were injected each afternoon for 12 weeks to compare the effectiveness of these two indoles in reducing T4 concentrations in hamsters maintained in the long photoperiod. Only the highest dose (200 micrograms) of 5-MT effectively suppressed T4 concentrations whereas all doses of MEL (greater than or equal to 5 micrograms) significantly reduced plasma T4. MEL or 5-MT (1-1,000 micrograms) were implanted in beeswax pellets in male hamsters exposed to short photoperiod (LD 10:14) to determine if either indole could prevent the short photoperiod-induced suppression of T4 (experiments 4 and 5). Regression and covariance analyses showed a significant log dose-related elevation of T4 in these hamsters, indicating equal potency of MEL and 5-MT in preventing the short photoperiod-induced suppression of T4. 5-MT given by either injection or implantation raised plasma triiodothyronine (T3) in female (experiment 2) but not in male hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

Concurrent determination of enzymatic activities and substrate concentrations in the melatonin synthetic pathway within the same rat pineal gland.

This study details the procedures involved in measuring a number of the componets of indole metabolism within the pineal gland. Tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid, N-acetyltransferase (NAT) activity, and hydroxyindole-O-methyltransferase activity were measured by a combination of three techniques, which included microassays, radioimmunoassay, and high-performance liquid chromatography. Determination of the melatonin synthetic pathway components within the same gland reduces the number of animals needed for studies and allows correlations between these constituents to be calculated. Very high degrees of correlation (r = 0.91-0.99) are seen between those compounds which exhibit significant rhythms (serotonin, NAT activity, N-acetylserotonin, and melatonin) when group means are compared. When correlations are calculated on a per-animal basis throughout the experiment, moderate to high degrees of correlation (r = 0.57-0.79) are found among those components that exhibit rhythms. However, when correlations are determined on a per-animal basis at each time point, no significant correlations are found. One hypothesis accounting for these differences may be that changes occur in indole metabolism within the same pineal gland over a period of a few minutes.

Relative efficacy of melatonin and 5-methoxytryptamine in terms of their antigonadotrophic and counterantigonadotrophic actions in male Syrian hamsters.

Antigonadotrophic and counterantigonadotrophic activities of melatonin and 5-methoxytryptamine (5-MT) were quantitatively compared in male Syrian hamsters. In long day conditions, the daily afternoon administration of either 5, 15, 25, 50, 100 or 200 micrograms melatonin induced testicular regression within 10 wk; under the same circumstances, only the 200-microgram dosage of 5-MT was able to suppress testicular weights. Thus, 5-MT appears to have about one-tenth the antigonadotrophic action of melatonin in the male Syrian hamster. In short days, the subcutaneous implantation of either 50 or 100 micrograms melatonin (every 2 wk in beeswax) prevented testicular regression whereas it required much larger doses of 5-MT (1 mg every 2 wk in beeswax) to achieve the same counterantigonadotrophic action. In terms of both their antigonadotrophic and counterantigonadotrophic effects, hamsters seem to be more sensitive to melatonin than to 5-MT.

The influence of different light irradiances on pineal N-acetyltransferase activity and melatonin levels in the cotton rat, Sigmodon hispidus.

Wild-captured cotton rats (Sigmodon hispidus) trapped and tested in September and October exhibited a rapid reduction in pineal N-acetyltransferase (NAT) activity and melatonin levels after exposure to a light irradiance of 300 microW/cm2 during the dark period. The half-time for the depression of both NAT and melatonin was on the order of 2 min. The exposure of cotton rats during darkness to much lower irradiances of light, i.e., 5.0, 0.04, 0.03 or 0.01 W/cm2, for 32 min also greatly diminished pineal NAT activity and radioimmunoassayable melatonin levels; however, a light irradiance of 0.005 microW/cm2 failed to significantly depress either the acetylating enzyme or the melatonin content of the pineal gland. The results show that the pineal gland of the wild-captured cotton rat, as judged by NAT activity and melatonin levels, is inhibited even by very low irradiances of light.