International Conference on Harmonization--critical discussion of the biotech 'Specifications' document.

Since 1990, the International Conference on Harmonization (ICH) has served as the primary medium for the development of consistent, harmonized and scientifically based international standards that keep abreast of the complexity of rapidly evolving technologies, and health, safety and commerce issues. Of the 45 guidance documents generated to date, influencing the conduct of drug development at various points during its continuum, six are dedicated to biotechnology. 'Specifications', the last in the series, was completed in 1999. It is fully complimentary to the other five guidance documents in the ICH biotechnology compendium. The process of establishing product specifications (principles and applications) is functionally couched within the multifaceted strategy of ensuring quality and consistency, and is proving to be a fundamental resource in crafting future harmonized documents such as the Common Technical Document.

FDA perspective on specifications for biotechnology products--from IND to PLA.

Quality standards are obligatory throughout development, approval and post-marketing phases of biotechnology-derived products, thus assuring product identity, purity, and potency/strength. The process of developing and setting specifications should be based on sound science and should represent a logical progression of actions based on the use of experiential data spanning manufacturing process validation, consistency in production, and characterization of relevant product properties/attributes, by multiple analytical means. This interactive process occurs in phases, varying in rigour. It is best described as encompassing a framework which starts with the implementation of realistic/practical operational quality limits, progressing to the establishment/adoption of more stringent specifications. The historical database is generated from preclinical, toxicology and early clinical lots. This supports the clinical development programme which, as it progresses, allows for further assay method validation/refinement, adoption/addition due to relevant or newly recognized product attributes or rejection due to irrelevance. In the next phase, (licensing/approval) specifications are set through extended experience and validation of both the preparative and analytical processes, to include availability of suitable reference standards and extensive product characterization throughout its proposed dating period. Subsequent to product approval, the incremental database of test results serves as a natural continuum for further evolving/refining specifications. While there is considerable latitude in the kinds of testing modalities finally adopted to establish product quality on a routine basis, for both drugs and drug products, it is important that the selection takes into consideration relevant (significant) product characteristics that appropriately reflect on identity, purity and potency.

Physiology of hemostasis.

The consequences of acute insults to the hemostatic system, whether congenital or acquired, frequently present a considerable challenge in diagnosis and therapy. Logical and effective management depends upon the proper identification of the hemostatic compartments involved; an appreciation for the considerably complex, delicately modulated interplays of various enzyme/inhibitor systems; and knowledge of the mechanism by which a variety of apparently unrelated disease processes precipitate sometimes catastrophic events--thrombosis/embolism/hemorrhage. We have attempted a logical review of basic mechanisms of hemostasis. The text is obligatorily brief, focusing on key elements of the biochemistry and physiology of the vessel wall, the platelet, and pertinent plasma factors. The section on plasma proteins pays particular attention to biocybernetic principles (positive/negative feedback loops) and to the interrelationship of enzyme systems involved in coagulation, fibrinolysis, kinin generation, and complement activation. No attempt was made to be encyclopedic. In the interest of brevity and clarity, the text has been limited to current concepts, with the reference material selected, whenever possible, in the form of review articles, volumes and monographs. We apologize for omissions. It is our belief that a working knowledge of basic mechanisms provides not only advantages in diagnostic/therapeutic management but also serves as a firm foundation for the development of novel diagnostic and therapeutic modalities.

Migration of dog polymorphonuclear neutrophilic leukocytes to formylated peptides.

Formylated peptides are potent stimulants of polymorphonuclear neutrophilic leukocyte (PMN) migration from species such as humans and rabbits. Interestingly, PMNs from dogs, cats, pigs and cows have been reported as refractory to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and generally are believed not to express formylpeptide receptors. Formylpeptides are a major component of conditioned media from E. coli cultures and believed to be a significant element in inflammatory responses elicited by E. coli. Our studies have found that E. coli filtrate was a potent stimulant of dog PMN migration. Inhibition of migration to E. coli filtrates by the antagonist t-botyloxycarbonyl-l-methionyl-l-leucyl-l-phenylalanine (t-boc-MLP) demonstrated that the migration was mediated through the formylated peptide receptor. Migration in response to peptides with higher affinity for the formylpeptide receptor than FMLP was further evidence for these receptors on the dog PMN. PMNs from dogs migrated in response to FMLP at high concentrations (100 microM); however, pretreatment with phorbol myristate acetate resulted in increased migration of dog PMNs in response to concentrations of FMLP as low as 1 pM. These results demonstrate that dog PMNs are responsive to formylpeptides and that these responses can be up-regulated by PMA. Thus PMNs from a species previously thought incapable of responding to formylpeptides can respond to formylpeptide analogs with high affinity for the receptor as well as be primed for enhanced migration to FMLP by PMA.

Examination of the role of the urokinase receptor in human colon cancer mediated laminin degradation.

The relevance of urokinase receptors to urokinase-mediated laminin degradation was investigated in cultured colon cancer. Six colon cancer cell lines degraded laminin in a plasminogen-dependent manner. The ability of the individual cell lines to cleave the glycoprotein correlated well (r2 = 0.9242) with the amount of urokinase recovered from the cell surface by a mild acid treatment. A radioreceptor assay indicated that colon cancer cells most active in degrading the laminin, possessed the largest number of urokinase receptors. Moreover, acid treatment which depletes the receptors of endogenous plasminogen activator augmented the specific binding of radioactive urokinase to the colon cancer cells by 12-200%. A cell line (HCT 116) which displayed 1.1 x 10(5) receptors/cell the majority of which were occupied with endogenous urokinase was selected and the effects of a urokinase receptor antagonist on laminin degradation determined. The peptide antagonist reduced laminin turnover by 60-80%. Morphological observations were consistent with these findings. Plasminogen-treated HCT 116 cells retracted from the culture dish and many cells were observed in the culture medium. This effect could be largely reversed by simultaneous treatment with the peptide antagonist. A poor correlation was found between laminin degradation and soluble urokinase (r2 = 0.1074). These data strongly argue for a role of the urokinase receptor in facilitating the action of the plasminogen activator in colon cancer.

Chronobiological evidence for an uncoupling of the Na,K-ATPase to aldosterone in normal renin hypertension.

The Na,K-ATPase activity of erythrocyte membranes is markedly increased in normal-renin essential hypertensives. A temporal shift of the chronobiology of the erythrocyte-membrane-bound Na,K-ATPase in these patients is described. The disorder causes a loss of synchronism between the circadian rhythms of aldosterone and Na,K-ATPase. Such uncoupling phenomenon may explain the inversion of the day/night sodium excretion ratio and other disturbances of sodium metabolism found in essential hypertensives.

Clinical use of biologicals produced in continuous cell lines.

Recent advances in biotechnology, namely DNA cloning and hybridoma formation, have greatly increased the availability of biologicals for clinical use. cDNA directed proteins and monoclonal antibodies may now be produced in sufficient quantities for wide-ranging experimental uses and, in some cases, licensed applications. Furthermore, novel uses of biological substances produced in minute amounts for normal homeostasis may now be broached using "pharmacological" doses. Biologicals produced in continuous cell lines have found application in wide-ranging fields. These include: hormone replacement, vaccines, immunosuppression and immunotherapy, and control of homeostasis. Initial efforts have been focused in oncology and infectious disease, including HIV-infection. Currently, therapies for a wide variety of conditions including thrombo-hemorrhagic syndromes, inborn errors of metabolism, cataracts, and arthritis are being developed. Conceivably, virtually every aspect of medicine, including diagnosis and surgery, will be affected by this revolutionary approach.

Modulation of the urokinase receptor in human colon cell lines by N,N-dimethylformamide.

The present study documents the effect of the planar, polar differentiation promoter N,N-dimethylformamide (DMF) on urokinase binding to colon carcinoma cells. Exposure of the colon carcinoma cell lines to the agent resulted in enhanced specific binding of radioactive urokinase to all cells tested. Insulin binding to the cells was, however, unaffected by DMF. A DMF exposure period of 45 h was required to observe maximum urokinase binding to two representative cell lines FET and RKO. Optimal stimulation of both cell lines occurred with 0.8% DMF. Scatchard analysis revealed the dissociation constants to be unchanged by the agent with the increased binding of radioactive plasminogen activator reflecting an up-regulation of binding sites. In this regard, the cell line RKO upon exposure to DMF, displayed approx. 700,000 receptors/cell, the highest value published, to date, for any cell line.

The gerontological decline of the renin-aldosterone system: a chronobiological approach extended to essential hypertension.

The circadian (about 24-hr) oscillating function of the renin-angiotensin-aldosterone system (RAAS) was investigated as a function of age in clinically healthy participants and in essential hypertensive patients. A peculiar age-related decline in the RAAS circadian mesor (rhythm-adjusted mean) and amplitude (variability from mesor) was found in the essential hypertensive patients. This finding suggests a nonphysiologic evolution in the tonic (24-hr mean level) as well as phasic (oscillating amplitude) circadian activity of the RAAS with increasing age. A relative hyperreninemic aldosteronism characterized the aged essential hypertensive patients.

Increased serum aldosterone in diabetic pregnancy.

Serum levels of aldosterone and cortisol were measured by radioimmunoassay in 15 patients with gestational diabetes, in 18 patients with Type 1 (insulin-dependent) diabetes, in 36 pregnant control women and in 10 non-pregnant control women. All subjects, on habitual sodium and potassium intake, were sampled in a supine position at 09.00 hours. Pregnant women were examined twice, during gestational week 32-34 and at delivery. Serum levels of aldosterone and cortisol were also measured in the umbilical cord blood of newborn babies of these diabetic and non-diabetic mothers. Serum levels of aldosterone in both gestational and Type 1 pregnant diabetic women were found to be consistently above the reference values of non-diabetic pregnant women. Abnormal serum levels of aldosterone were also observed in newborn infants of diabetic mothers. In contrast, serum levels of cortisol were not increased.

Effects of a mild and prolonged restriction in sodium or food intake on the circadian rhythm of aldosterone and related variables.

The effect of a mild reduction in dietary sodium intake (-30 mEq/24 hr) and body weight (-2 kg/2 months) on circadian rhythms of urinary aldosterone (UA), sodium (UNa), potassium (UK), creatinine (UC) and volume (UV) have been investigated in nine clinically healthy subjects. The mild reduction in dietary sodium is associated with: (1) a decrease in the 24-hr excretion rate of UNa, UK and UV, and an increased mesor of UA and UC; (2) a lowered extent of the circadian variation for UNa, UK, UV and a greater amplitude for UA and UC (3) a later crest in the temporal phase for UK, UA, UC, an earlier phasic wave for UNa. The mild reduction in calorie intake resulting in a body weight loss is associated with a more pronounced decrease in the 24-hr excretion rate of UNa and UK, and in the extent of circadian fluctuation for UNa. Peculiar events are: (1) the decreased 24-hr excretion rate for UA, and the increased mesor for UV; (2) the extent variability increased for UV, decreased for UC. Such effect may have a practical resonance for heuristic physiology since the role of dietary sodium and food intake has been better clarified. Dietary sodium and food can be regarded as 'chronomodulatory agents' for the adrenal cortex since their adrenotropic influence is extended to the tonic as well as phasic secretion of aldosterone.

Occurrence of a circadian rhythmicity for blood pressure in patients with circulatory shock.

Ten patients hemodynamically in shock were monitored for blood pressure along the 24-hr span while under antishock therapy. Time-qualified data were analyzed by means of the cosinor procedure. The fit of a 24-hr cosine function was able to reject the null hypothesis of amplitude = O in the majority of patients under intensive care. The blood pressure circadian rhythm was found to be independent of whether the patient was responsive or refractory to therapy. This phenomenon demonstrates that the maintenance of a blood pressure circadian rhythm is a characteristic intrinsic to hemodynamic shock even if blood pressure falls to very low values along the time scale.

Postural effects on the circadian rhythm of blood pressure and heart rate in young and elderly subjects.

This investigation compares the effects of changes in activity-rest patterns on the 24-hr rhythms of blood pressure and heart rate in elderly and young subjects. Blood pressure and heart rate were monitored by means of noninvasive, automatic, quasiportable recording equipment. The subjects either rested both diurnally and nocturnally or were active diurnally and rested nocturnally. Time-qualified data were analyzed for circadian rhythmicity by means of the cosinor procedure. In both activity-rest patterns, elderly subjects showed a dissociation between circadian rhythms of blood pressure and heart rate due to a peculiar shift of acrophases. Because of the particular timing of the acrophase, elderly subjects differed from young individuals in the overall adaptation of the blood pressure and heart rate 24-hr patterns to upright posture and physical activity. Of particular interest is the reduced amplitude in the circadian rhythm of blood pressure, which might contribute to the compromised orthostatic tolerance of older people.

Internal desynchronization between circadian rhythms of plasma aldosterone and erythrocyte membrane-bound Na/K-ATPase.

Normal-renin essential mesor hypertensives are characterized by a consistent increase in erythrocyte membrane-bound Na/K-ATPase activity. Low-renin essential hypertensives exhibit, in contrast, a lower activity in Na/K-ATPase of cell membranes. This study documents a third disorder characterized by a temporal shift in the rhythmic activity of the erythrocyte membrane-bound Na/K-ATPase in normal-renin hypertensives. The disorder causes the synchronism with the aldosterone circadian rhythm to be invariably lost. The uncoupling phenomenon could be invoked to explain the inversion in the day-night sodium excretion rate found in essential hypertensives. In addition, it suggests that the circadian rhythm in Na/K-ATPase is under the control of cycling factors other than aldosterone.

Tumor cell autocrine motility factor.

A cell motility-stimulating factor has been isolated, purified, and partially characterized from the serum-free conditioned medium of human A2058 melanoma cells. We term this activity "autocrine motility factor" (AMF). AMF has the properties of a protein with an estimated size of 55 kDa. At concentrations of 10 nM or less, AMF stimulated the random or directed motility of the producer cells. However, AMF is not an attractant for neutrophils. Amino acid analysis of the purified AMF protein revealed a high content of serine, glycine, glutamic acid, and aspartic acid residues. The activity of AMF was not replaced or blocked by known growth factors such as epidermal growth factor or type beta transforming growth factor. Mechanistic studies showed that AMF stimulated the incorporation of [3H]methyl into cell membrane phospholipids after incubation with [methyl-3H]methionine with a sustained increase in the methylation of phosphatidyldimethylethanolamine to phosphatidylcholine. In contrast, AMF did not affect the incorporation of [1,2-14C]choline into phosphatidylcholine. AMF was produced in large amounts by three different clones of ras oncogene-transfected metastatic NIH 3T3 cells but not by the nontransformed parental cells. AMF may play a major role in the local invasive behavior of tumor cells and may also facilitate the concerted invasion by groups of tumor cells.