Modernizing methodology for the WHO assessment of substances for the international drug control conventions.

BACKGROUND: The WHO Executive Board revised the guidance that governs the procedures for the WHO review of psychoactive substances for international drug control in 2010. To meet the standards defined in these guidelines, the current evaluation methodology at WHO must be an evidence-based assessment. METHODS: We describe the history of substance evaluation from 1912 to the present and the development of the evaluation methods over time including a description of the current assessment system, using reports from WHO and its predecessor, the League of Nations. Furthermore, we describe the current review system. RESULTS: We found that some substances under international control were never reviewed; other substances were reviewed decades ago. CONCLUSIONS: We argue that assessments do not have unlimited validity, and therefore, substances need to be re-assessed periodically, as already recommended by the Expert Committee on Drug Dependence in 1982. We propose that the evaluation time be shortened; that the influence of the route of administration and/or dosage form of the preparation is considered in the evaluation; and we recommend studying national and regional assessment systems and adopting their best practices. With this article, we make a case for the inclusion of systematic review and other methods of comprehensive analysis of substance evaluation to arrive at a process of equal rigour and quality as already applied by WHO for the development of treatment guidelines.

Process characterization for metal-affinity chromatography of an Fc fusion protein: a design-of-experiments approach.

The utility of a design-of-experiments approach was investigated for process characterization of a metal-affinity chromatographic purification process for an Fc fusion protein. This approach gave a better understanding of some of the key process variables as well as robustness for this step in the purification process. Single-variable experiments were employed to screen some of the potentially important variables in this step. Ranges for these variables were set based on prior experience in clinical manufacturing with similar processes. Following these experiments, a fractional factorial study was employed to further investigate the most important variables and their interactions. Key operational variables that had an impact on step yield and eluate purity were identified. In addition, the study helped identify a worst-case scenario for the step purity and helped assure that the rest of the process would successfully purify the product. This paper demonstrates the utility of a design-of-experiments approach for the characterization and validation of process chromatography steps in downstream processing. In addition, this study emphasizes the utility of robustness studies early in process development and establishes a strategy for future robustness studies.

An early developmental phase of pp60c-src expression in the neural ectoderm.

The expression of the normal cellular src protein (pp60c-src) was investigated in the early chick embryo during gastrulation and neurulation by immunoperoxidase staining using antisera, raised against bacterially expressed pp60v-src, that recognizes pp60c-src specifically in normal cells. During gastrulation pp60c-src immunoreactivity appeared primarily in the neural ectoderm and was much less prominent in the mesoderm, endoderm, and nonneural ectoderm. During neurulation pp60c-src immunoreactivity began to disappear from the wall of the closing neural tube so that by the completion of neural tube closure no specific pp60c-src immunoreactivity appeared in any of the neuroepithelial cells composing the neural tube. These studies reveal a developmental phase of pp60c-src expression even earlier than reported previously, when neuroepithelial cells of later embryos undergo terminal neuronal differentiation. These findings raise the possibility that pp60c-src may mediate two different differentiation signals in the neuronal lineage.

pp60c-src is expressed in human fetal and adult brain.

Human cells contain a tyrosine-specific protein kinase, pp60c-src, that is highly homologous to the oncogene product, pp60v-src, from Rous sarcoma virus but is of unknown function. The expression of human pp60c-src was examined in tissues obtained from human adults and fetuses of 20-32 weeks' gestational age. pp60c-src was quantitated in tissue extracts by measurement of its protein kinase activity by the use of the immune complex protein kinase assay. Brain showed the highest levels of pp60c-src protein kinase activity, but all other human tissues examined had significant levels. Fetal tissues, including brain, showed three- to eight-fold higher levels of pp60c-src kinase activity than the corresponding adult tissues. pp60c-src kinase was found to be uniformly distributed in the adult brain; frontal, occipital, and parietal cortex, and cerebellum expressed equivalent amounts of pp60c-src kinase activity. The protein kinase activity in human tissues exhibited properties characteristic of pp60c-src in other species, namely, tyrosine-specific phosphorylation of specific antibody heavy chains, autophosphorylation of a 60,000 Mr protein following immunoprecipitation with a monoclonal antibody specific for pp60src, and sensitivity to inhibition by P1,P4-di(adenosine-5')tetraphosphate. The high levels of human pp60c-src in fetal tissues, particularly in brain, suggest a possible function in developmental processes.

A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver.

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single Mr 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthase, microtubule-associated proteins, tubulin, alpha-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.

pp60c-src Kinase is in chick and human embryonic tissues.

The normal cellular protein pp60c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60c-src distributed throughout the neural tube and heart. The distribution of pp60c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60c-src kinase. The results of our studies suggest that pp60c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.

pp60c-src is developmentally regulated in the neural retina.

We have localized normal cellular pp60c-src in the developing chick neural retina by immunocytochemical staining using antisera raised against bacterially expressed pp60v-src, the src gene product of Rous sarcoma virus. pp60c-src was expressed in developing retinal neurons at the onset of differentiation. Expression of pp60c-src persisted in mature neuronal cells that were postmitotic, fully differentiated, and functional. pp60c-src immunoreactivity was localized within processes and cell bodies of ganglion neurons, processes of rods and cones, and in some but not all neurons of the inner nuclear layer. Protein kinase assays and Western transfer analyses identified the immunoreactive protein as pp60c-src, and confirmed that its expression occurs at the time the first neuronal cells in the retina differentiate. We conclude from these studies that pp60c-src is the product of a developmentally regulated gene that is more important in neuronal differentiation or function than cell proliferation.

Differential inhibition of cellular and viral pp60src kinase by P1,P4-di(adenosine-5')tetraphosphate.

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.

Effect of environmental hydrogen ion concentration on regulation of insulin receptors in cultured R3230AC mammary carcinoma cells.

Insulin binding and responsiveness in primary cultures of R3230AC rat mammary tumor cells were studied as a function of the hydrogen ion concentration of the culture medium. When insulin binding was assayed at pH 7.4, cultures that were maintained for a sufficient length of time in acidic medium demonstrated a significant increase in insulin receptor concentration compared to control cultures. The increase in insulin binding, which occurred as cultures approached confluency, was attributed to the increased acidity rather than nutrient depletion of the culture medium. In contrast, maximum binding was observed when the pH of the assay buffer was above 8.0, independent of the culture conditions. Binding of Concanavalin A, reflecting more generalized cell surface glycoproteins, decreased as cultures approached confluency, but was unaffected by the pH of either the culture medium or the assay buffer. The effect of pH on insulin responsiveness was studied. Insulin receptors generated by acidic culture conditions demonstrated insulin-induced down-regulation. Regardless of the pH environment, all cells demonstrated the same amount of insulin binding after exposure to 10(-6) M insulin. Under the in vitro conditions employed, cultured cells did not demonstrate a significant response to added insulin by alteration in growth, substrate transport, or incorporation of precursors into macromolecules, although the basal rates of these parameters were lower in cells maintained in acidic pH environments. The data presented indicate the necessity of considering the pH of the culture medium in studies of receptor regulation. It is possible that tumor cells, due to increased lactic acid production, may be especially prone to these changes.

Down-regulation of insulin receptors in primary cultures of R3230 AC rat mammary adenocarcinoma cells.

Binding of insulin and Concanavalin A to primary cell cultures of the R3230AC rat mammary adenocarcinoma was studied as a function of time in culture. As the culture became confluent, the amount of insulin binding per cell increased with culture time and reached a plateau, whereas the binding of Con A to surface glycoproteins decreased to 50% of the initial value. Exposure of confluent cultures to insulin at 37 C resulted in down-regulation of the cell surface insulin receptors. The decrease in insulin binding was related to the ambient insulin concentration and the decreased numbers of receptors per cell with no apparent alteration in their affinity. The maximal decrease in receptor number was 60-70%. Cell cultures degraded significant amounts of insulin at 37 C, but the addition of bacitracin to the culture medium decreased the amount of degradation and increased the extent of down-regulation at each insulin concentration. Porcine proinsulin was less effective than insulin in competing with 125I-labeled insulin and inducing receptor down-regulation. Down-regulation of insulin receptors did not require protein synthesis. The rate of insulin-induced receptor loss was much faster than the decrease in insulin binding due to inhibition of protein synthesis by cyclohexamide. The estimated half-life of the insulin receptor was 10.5 h. Down-regulation of insulin receptors was reversible; regeneration of receptors to 50% of control levels occurred approximately 9.6 h after the removal of insulin and required protein synthesis. These results indicate that these mammary tumor cells retain the ability to regulate their insulin receptors.

The hormonal regulation and interaction of concanavalin A and insulin binding in R3230AC mammary adenocarcinoma cells.

To investigate the effects of concanavalin A on insulin binding to R3230AC mammary carcinomas, initial experiments were performed to characterize binding of concanavalin A. Concanavalin A binding was found to be specific and saturable. Equilibrium binding experiments demonstrated that addition of low concentrations of concanavalin A enhanced the binding of [3H]concanavalin A, suggestive of positively cooperative interactions. Binding of concanavalin A was responsive to hormonal alterations; tumor cells from diabetic rats showed enhanced binding of concanavalin A and insulin compared to cells from intact rats and administration of insulin to diabetic rats returned concanavalin A and insulin binding to levels seen in controls. Incubation of tumor cells with concanavalin A prior to addition of 125I-labelled insulin resulted in a reduction of insulin-binding capacity; succinyl-concanavalin A did not affect binding of insulin. The present inhibition of insulin binding by concanavalin A was highest at the lower insulin concentrations, providing a linearized Scatchard plot that yielded a calculated Kd value comparable to the low-affinity portion of the curvilinear Scatchard plot for insulin binding. The dissociation rate of bound insulin depended on receptor occupancy. Addition of concanavalin A after insulin binding reached equilibrium resulted in increased insulin binding at higher hormone concentrations, decreased rates of dissociation of insulin and a loss of the correlation between receptor occupancy and dissociation rates. Concanavalin A alone demonstrated an insulin-like effect on glucose transport, which in these tumor cells represents a decrease in transport of 3-O-methylglucose. These results suggest that binding of both concanavalin A and insulin to cells from this hormonally responsive neoplasm is under insulin regulation and demonstrates similar characteristics to those reported for a variety of normal cells. Furthermore, the interaction between concanavalin A and te cell membranes affects the affinity of the insulin receptor for insulin and appears to decrease the observed negative cooperativity.