On the Shelf Life of Pharmaceutical Products

This article proposes new terminology that distinguishes between different concepts involved in the discussion of the shelf life of pharmaceutical products. Such comprehensive and common language is currently lacking from various guidelines, which confuses implementation and impedes comparisons of different methodologies. The five new terms that are necessary for a coherent discussion of shelf life are: true shelf life, estimated shelf life, supported shelf life, maximum shelf life, and labeled shelf life. These concepts are already in use, but not named as such. The article discusses various levels of "product" on which different stakeholders tend to focus (e.g., a single-dosage unit, a batch, a production process, etc.). The article also highlights a key missing element in the discussion of shelf life-a Quality Statement, which defines the quality standard for all key stakeholders. Arguments are presented that for regulatory and statistical reasons the true product shelf life should be defined in terms of a suitably small quantile (e.g., fifth) of the distribution of batch shelf lives. The choice of quantile translates to an upper bound on the probability that a randomly selected batch will be nonconforming when tested at the storage time defined by the labeled shelf life. For this strategy, a random-batch model is required. This approach, unlike a fixed-batch model, allows estimation of both within- and between-batch variability, and allows inferences to be made about the entire production process. This work was conducted by the Stability Shelf Life Working Group of the Product Quality Research Institute.     

FGF-2 release from the lens capsule by MMP-2 maintains lens epithelial cell viability

The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.     

Microzooplankton grazing and the control of phytoplankton biomass in the Suwannee River estuary,USA

Microzooplankton grazing can have significant impacts on the distribution and abundance of phytoplankton, thereby influencing the frequency and duration of algae blooms. Observations of high ciliate abundances in the Suwannee River estuary, Florida, suggest a significant potential for top-down pressure on the phytoplankton community by microzooplankton. We examined the composition of the microzooplankton and determined grazing mortality losses for phytoplankton within the Suwannee River estuary from 2001 to 2002. Our results indicated grazing mortality rates of 1.4 d⁻¹, equivalent to a loss of up to 76% of phytoplankton standing crop and up to 83% of total daily primary production. The microzooplankton community was primarily composed of ciliates, dinoflagellates, and copepod nauplii. The densities of ciliates in the estuary were comparable to densities reported in highly eutrophic ecosystems (9,400–72,800 ciliates l⁻¹). Grazing pressure on small phytoplankton may be further enhanced because ciliates and small dinoflagellates have growth rates similar to those of phytoplankton, and therefore can keep up with surges in abundance. Handling editor: Judit Padisak     

Extrinsic Mortality Effects on Reproductive Strategies in a Caribbean Community

Extrinsic mortality is a key influence on organisms’ life history strategies, especially on age at maturity. This historical longitudinal study of 125 women in rural Domenica examines effects of extrinsic mortality on human age at maturity and pace of reproduction. Extrinsic mortality is indicated by local population infant mortality rates during infancy and at maturity between the years 1925 and 2000. Extrinsic mortality shows effects on age at first birth and pace of reproduction among these women. Parish death records show huge historical variation in age-specific mortality rates. The infant mortality rate (IMR) in the early 1920s was low, increased dramatically beginning in 1929, and reached a maximum in the 1950s, at which point IMR declined steadily to its present low rate. The mortality rate early in life showed a quadratic association with age at first birth. Women who experienced conditions of low IMR early in life reproduced relatively late in life. Those born into moderately high levels of infant mortality tended to reproduce earlier than those born at low levels. At very high infant mortality levels early in life, women went on to delay reproduction until relatively late, possibly as a result of somatic depletion and energetic stress associated with the conditions that lead to high IMR. Population mortality rates at age of maturity also showed a quadratic association with age at first birth. The pace of reproduction, estimated as number of surviving offspring controlled for maternal age, showed a similar quadratic effect. There were complex interactions between population mortality rates in infancy and at maturity. When extrinsic mortality was high during infancy, extrinsic mortality later in life had little effect on timing of first birth. When extrinsic mortality was low to moderate in infancy, extrinsic mortality later in life had significant effects on adult reproduction. I speculate that these effects are mediated through development of personality facets associated with reproduction.     

Regulation of the mitochondrial matrix volume in vivo and in vitro. The role of calcium.

The ability of alpha-adrenergic agonists and vasopressin to increase the mitochondrial volume in hepatocytes is dependent on the presence of extracellular Ca2+. Addition of Ca2+ to hormone-treated cells incubated in the absence of Ca2+ initiates mitochondrial swelling. In the presence of extracellular Ca2+, A23187 (7.5 microM) induces mitochondrial swelling and stimulates gluconeogenesis from L-lactate. Isolated liver mitochondria incubated in KCl medium in the presence of 2.5 mM-phosphate undergo energy-dependent swelling, which is associated with electrogenic K+ uptake and reaches an equilibrium when the volume has increased to about 1.3-1.5 microliter/mg of protein. This K+-dependent swelling is stimulated by the presence of 0.3-1.0 microM-Ca2+, leading to an increase in matrix volume at equilibrium that is dependent on [Ca2+]. Ca2+-activated K+-dependent swelling requires phosphate and shows a strong preference for K+ over Na+, Li+ or choline. It is not associated with either uncoupling of mitochondria or any non-specific permeability changes and cannot be produced by Ba2+, Mn2+ or Sr2+. Ca2+-activated K+-dependent swelling is not prevented by any known inhibitors of plasma-membrane ion-transport systems, nor by inhibitors of mitochondrial phospholipase A2. Swelling is inhibited by 65% and 35% by 1 mM-ATP and 100 microM-quinine respectively. The effect of Ca2+ is blocked by Ruthenium Red (5 micrograms/ml) at low [Ca2+]. Spermine (0.25 mM) enhanced the swelling seen on addition of Ca2+, correlating with its ability to increase Ca2+ uptake into the mitochondria as measured by using Arsenazo-III. Mitochondria derived from rats treated with glucagon showed less swelling than did control mitochondria. In the presence of Ruthenium Red and higher [Ca2+], the mitochondria from hormone-treated animals showed greater swelling than did control mitochondria. These data imply that an increase in intramitochondrial [Ca2+] can increase the electrogenic flux of K+ into mitochondria by an unknown mechanism and thereby cause swelling. It is proposed that this is the mechanism by which alpha-agonists and vasopressin cause an increase in mitochondrial volume in situ.     

Influence of Benzyladenine, Leaf Darkening, and Ringing on Movement of C-labeled Assimilates Into Expanded Leaves of Vitis vinifera L

Leaves of Vitis vinifera L., nearly fully expanded, imported only trace amounts of ¹⁴C following assimilation of ¹⁴CO₂ by a lower leaf on the same shoot, but benzyladenine (BA) application at 4.4 × 10⁻³m caused a marked increase in the movement of ¹⁴C into these leaves. Older leaves near the shoot base were less responsive; BA treatment alone had little effect on import of labeled assimilates from adjacent leaves but when the BA-treated leaves were darkened there was an increased import of labeled materials. When these 2 treatments were combined and applied to leaves on shoots with ringed bases, relatively high levels of radioactivity were detected in the BA-treated leaves but under these conditions darkening, without the application of BA, also resulted in an increased import of ¹⁴C. Accumulation of imported ¹⁴C was found to be restricted to the area of the leaf blade treated with BA. Separation of labeled compounds in ethanol extracts of treated leaves showed a lower percentage of radioactivity present in the sugar fraction from BA-treated leaves and an increased percentage present in the amino acid fraction.     

Partial Purification of a Membrane-Associated Deoxyribonucleic Acid Complex from Mycoplasma gallisepticum

A Mycoplasma gallisepticum subcellular fraction (P2), which contains the deoxyribonucleic acid replication complex, can be isolated by differential centrifugation of freeze-thaw-lysed cells. The nascent deoxyribonucleic acid is released from P2 by Lubrol-WX, sodium dodecyl sulfate, Pronase, and deoxyribonuclease, but not by saponin, ribonuclease, phospholipase C, or high-frequency sonic treatment. Sonic treatment further fractionates the cell ghost and allows partial purification, on sucrose density gradients, of a deoxyribonucleic acid replication complex attached to the cells' polar membrane-bleb-infrableb structures.     

The concept of mRNA abundance classes: a critical reevaluation.

Derivative plots have been constructed for hybridization reactions between polysomal poly(A)-containing RNA and oligo(dT)-primed cDNA. In one method the derivative was calculated directly from the data, and in the other, from a non-linear least squares fit using 9-10 ideal components. In some cases these methods yield very similar results and strongly suggest that the hybridization data support discrete components. Reactions with two and four major components indicate that the often-reported three abundance class model is only one of several possibilities for eukaryotic cells. In other situations neither method strongly suggests the presence of discrete components (in one case even after enrichment of the cDNA population by kinetic fractionation), implying that the components are closely spaced or that the entire mRNA population of those cells may not exist as discrete abundance classes. The universal occurrence of discrete abundance classes should be critically reexamined.     

Sodium-gradient-stimulated transport of l-alanine by plasma-membrane vesicles isolated from liver parenchymal cells of fed and starved rats. Crucial role of the adrenal glucocorticoids

The ability of liver efficiently to take up amino acids, particularly l-alanine, during starvation was studied in a cell-free system by isolating plasma-membrane vesicles in a transport-competent state from rat liver parenchymal cells. These membrane vesicles have the capacity to accumulate l-alanine against an apparent concentration gradient when exposed to an artificial and transient transmembrane Na⁺ gradient (extravesicular Na⁺ concentration greater than inside). The rate of accumulation of l-alanine is dependent on the plasma-membrane vesicle concentration, and the steady-state concentration attained is inversely related to the osmolarity of the medium. The Na⁺-mediated stimulation is not exhibited if the membrane vesicles are pre-equilibrated with NaCl, if K⁺ or Li⁺ are substituted for Na⁺, or if SO₄²⁻ replaces Cl⁻ as the counterion. The apparent active transport of l-alanine into the membrane vesicles appears to occur by an electrogenic mechanism: (1) the use of NaSCN significantly heightens the early concentrative phase of transport when compared with the effect of NaCl; (2) an enhanced active transport is also observed when a valinomycin-induced K⁺ efflux occurs concomitant with Na⁺ and l-alanine influx. Plasma-membrane vesicles isolated from liver parenchymal cells of a 24 h-starved rat exhibit an initial l-alanine transport rate that is 3–4 times that for membrane vesicles derived from a fed animal. The increased rate of l-alanine transport by plasma-membrane vesicles from starved animals can be obliterated by adrenalectomy and restored by administration of glucocorticoid. These results establish that stimulation of the gluconeogenic pathway by starvation involves a plasma-membrane-localized change affecting l-alanine transport which is regulated in part by the glucocorticoid hormones.