Bioequivalence of racemic drugs.

Although pharmacokinetic and pharmacodynamic differences between the enantiomers of a chiral drug have been known or suspected for many years, racemate drugs have frequently been developed and approved without clinical pharmacologic consideration of their chiral components. In the late 1970s, the technology to isolate, manufacture, and detect pure enantiomers of racemate drugs became generally available. This availability has created new demands on both pharmaceutical firms and regulatory agencies. To prepare for this new technology, the Center for Drug Evaluation and Research at the Food and Drug Administration is formulating a policy statement to guide evaluation of new chiral drugs. At this time, it appears that whatever new policies are developed will not necessarily be applied retroactively to previously approved racemate drugs. Additional policies to guide the development and approval of generic and OTC chiral drugs may be required. In the Office of Generic Drugs in the Center, abbreviated new drug or antibiotic applications are approved on the basis of adequate chemistry, manufacturing, and control procedures and comparative pharmacokinetics (bioequivalence). The generic drug must be a racemate or single enantiomer if the corresponding innovator drug is a racemate or single enantiomer respectively. Whether a generic firm will be required to provide bioequivalence information on enantiomers of a racemate is determined on a case-by-case basis. Although it might be claimed that a generic drug product should be required only to undergo the same general kind of pharmaceutical evaluation as did the innovator, there may be instances when the approval of a generic drug or antibiotic will require measurement of specific enantiomers of a chiral drug.

Degradation and internalization of bound insulin in human erythrocytes.

Internalization and degradation of insulin by human erythrocytes were studied. Erythrocytes were incubated with 125I-insulin at 4 degrees C, 15 degrees C, and 37 degrees C for varying time intervals. These erythrocytes were then subjected to a low pH wash to release bound insulin followed by TCA precipitation. After 4, 22, and 24 hours of insulin binding at 4 degrees C, 92 to 95% of the bound 125I-insulin was dissociable and 92 to 98% of the extractable insulin was undegraded. After 3.5 hours of incubation at 15 degrees, 82% of the bound insulin was dissociable and 60% of this was intact. However, after 60, 90, 120, and 180 minutes of incubation at 37 degrees C, only 42, 34, 24, and 37%, respectively, of the bound insulin was dissociable. The undissociated insulin in the 37 degrees C studies was considered to be intracellular. With increasing time of incubation at 37 degrees C, the extractability of cell bound insulin and the proportion of undegraded dissociable insulin were decreased. When 125I-insulin binding was 95% blocked by preincubating the erythrocytes with anti-insulin receptor antibody, 95% of the degradation of 125I-insulin was also blocked. These studies indicate that mature human erythrocytes degrade internalized insulin and this process is time, temperature, and insulin receptor dependent.

A simpler procedure to study sodium-22 uptake (ouabain insensitive) in human erythrocytes.

We report a modification of the technique of Mahoney et al. (Blood 1982; 59: 439) for the determination of sodium-22 (22Na+) uptake in human erythrocytes. This modification facilitates the separation of 22Na+ taken up by erythrocytes from the free 22Na+ in the buffer by the addition of dibutyl phthalate, which forms an immiscible layer between the two. To further improve the sensitivity of 22Na+ uptake, we incubated a range of known numbers of erythrocytes with 22Na+ as opposed to the single cell suspension of known hematocrit used in Mahoney's et al. procedure (1). Erythrocytes are incubated in KCI buffer containing 2627 Bq (0.071 microCi) 22Na+ in a total volume of 0.5 mL for 0.5 h at 37 degrees C. Incubation is terminated by placing the tubes in ice for 10 min and the amount of 22Na+ taken up by the erythrocytes determined. We observe a linear relationship between erythrocyte concentrations (0.5 to 2.5 X 10(9) cells/mL) and percent uptake of 22Na+ (0.37 +/- 0.06 (1 SD) to 1.85 +/- 0.27 (1 SD) of the total 22Na+, respectively). The procedure is simple and sensitive, and can be used in clinical laboratories for the routine evaluation of 22Na+ uptake in erythrocytes.

Characterization of an intracellular insulin-degrading enzyme in human erythrocytes.

Using conventional techniques of ammonium sulfate fractionation and Sephadex gel column chromatography, insulin-degrading enzyme was partially purified from lysate of human erythrocytes. The enzymatic activity was measured by the trichloroacetic acid precipitation method. Compared to trypsin, the enzyme was highly specific for insulin. The apparent molecular weight of the enzyme was 160,000 Da, the optimum pH was the 7.4 to 7.8 range, and the Km value for insulin for the partially purified enzyme was 162 nM. Bacitracin and N-ethylmaleimide were potent inhibitors, while chloroquine, ethylenediaminetetraacetate, antipain, and soybean trypsin inhibitor failed to inhibit the activity of the enzyme. Like most nucleated cells, human erythrocytes not only have the membranal insulin receptors, but also possess the cytosolic specific insulin-degrading enzyme. Insulin internalization and degradation are shown to be due to the receptor and the enzyme acting in concert as in many nucleated cells. Anucleated erythrocytes have both these entities for possible internalization and degradation of insulin.

Peroxidases induced in rat uterus by estrogen administration. II. Cytochemical and biochemical heterogeneity.

In estrogen and diethylstilbestrol-treated rats, uterine peroxidases originate from two sources, the infiltrating eosinophils (exogenous) and the uterine tissue itself (endogenous). The study reported here distinguished the exogenous peroxidases by biochemical means and by cytochemistry. Eosinophil peroxidases are confined to the stromal and myometrial regions and appear simultaneously with endogenous peroxidases. At 48 h after estrogen-administration, the clear uterine luminal washings contain five peroxidase isoforms; this increases to 7-15 isozymes by 72 h. Uterine fluid peroxidase isozymes are acidic proteins with pI values ranging from pH 4.0 - 7.2, while the principal eosinophil peroxidase is a basic protein with a pI value ranging from pH 8.0-8.9. Eosinophil peroxidase is electrophoretically demonstrable only in the presence of the cationic detergent cetyltrimethyl ammonium bromide (CTAB) and has a spectrophotometric optimum of pH 4.4. In contrast, uterine fluid peroxidases have a pH optimum of 7.2. and no requirement for CTAB. Uterine tissue peroxidase extracted in the presence of Ca2+, showed a minor electrophoretic peroxidase band in the acidic pH range; however, a CTAB-activated peroxidase similar to the principal eosinophil peroxidase appeared as a basic protein. The data strongly suggest that uterine fluid peroxidases are estrogen-induced peroxidases (EIP) distinct from the eosinophil peroxidases that are largely restricted to the stromal compartment. This conclusion is supported by cytochemical studies that show two eosinophilperoxidases. The one shown by DAB was resistant to cyanide whereas the one shown by PPD/PC was inhibited by cyanide. A uterine tissue peroxidase, which was demonstrated only in stromal cells by the DAB medium, was more sensitive to cyanide than the eosinophil peroxidase shown by DAB.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat erythrocyte insulin receptors: radioreceptor assay and characterization.

Highly specific insulin receptors have been identified on the rat erythrocyte. A radioreceptor assay for the evaluation of these receptors has been developed, and the characteristics of these receptors have been investigated. Insulin receptor binding on the rat erythrocytes was found to be dependent on pH, temperature, time, and ionic strength. When incubated for 3½ hours at 15° C, 5.0 × 10(9) erythrocytes/mL from each of 10 rats were found to bind specifically 7.54 percent (±0.15 SEM) of 40 pg of (125)I-insulin. Specific binding was found to be a function of cell concentration. The pH optima for insulin binding were found to be 7.4 and 7.0 in the absence of cations. The presence of cations not only shifted pH optimum to 7.4 from 7.0, but also increased specific insulin binding.These observations suggest the stabilization of negatively charged groups on ligand and receptor, as well as providing a suitable ionic environment for the hormone-receptor interaction. Based on the resistance of rat erythrocytes to the pH of the external buffer, a simple method for determining the internal pH of rat red- blood cells is described. Scatchard analyses of insulin-binding data yielded curvilinear plots, and the number of receptor sites per cell was found to be 762 (±12.1 SD), as opposed to the large variation (410 ± 260 SD) in normal humans. The rat erythrocytes may serve as a useful, precise, sensitive, and efficient model system for future erythrocytic-receptor studies that would be difficult to obtain from human subjects.

Metabolism of insulin in erythrocytes from renal failure patients on maintenance hemodialysis.

Glucose tolerance does not improve to the normal level after dialysis; however, our studies showed that the insulin receptor binding to erythrocytes of nondiabetic patients with chronic renal failure (CRF) on hemodialysis was more than that in normal subjects. To understand this apparent anomaly in insulin receptor action and glucose metabolism, we investigated insulin degradation-a postreceptor event of insulin binding-in erythrocytes from CRF patients and compared it with that of normal subjects. We studied insulin degradation by erythrocytes from each of eight CRF patients and five normal subjects. The average hyperbolic insulin degradation curve for the CRF patients showed lower activity and a right-handed shift compared to the curve for the normal subjects. The average maximum degradation of insulin in the CRF patients was significantly lower than that of normal subjects. The number of erythrocytes required to produce 50% of maximum insulin degradation was significantly greater in these patients than that in the normal subjects. Furthermore, a linear correlation was observed between the duration of dialysis and maximum percent of insulin degradation in the CRF patients. Clinical implications of these findings are unclear at the present time. However, the insulin-degrading activity in erythrocytes may be reflective of that in other body tissues.

Effect of storage on insulin receptor binding in human erythrocytes.

The authors established the specificity, reliability, and precision of human erythrocyte insulin radioreceptor assay. On the basis of insulin binding, cell viability, and degree of hemolysis, heparin sodium was found to be a more suitable anticoagulant than sodium fluoride, ethylenediaminetetraacetic acid, sodium oxalate, or sodium citrate. In two sets of experiments carried out at 4°C and 23°C, human erythrocytes were stored as whole blood or isolated erythrocytes suspended in Tris-{4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid} buffer. The effect of storage under these conditions was evaluated by erythrocytespecific insulin binding. Human erythrocytes can be stored for 72 hours at 4°C without any change in insulin binding, insulin receptor sites per cell, or average affinity constant at the empty sites. Isolated erythrocytes can also be stored in plasma for 72 hours or in buffer G for 24 hours at 4°C without any change in insulin binding. It is not advisable to store human erythrocytes in plasma or as whole blood for more than 24 hours at 23°. These findings are useful in preserving insulin receptor activity when storage of erythrocytes is unavoidable.

Insulin receptor characteristics of erythrocytes from human newborns.

Erythrocytes from human newborns were observed to have specific insulin receptors. The characteristics of these receptors were similar to those of the normal adult subjects. An observed slight increase in R(o) and a decrease in K̄(e) of insulin receptors in erythrocytes may be speculated to facilitate the transfer of insulin from the fetal erythrocytes to other rapidly growing fetal tissues at a rate faster than that present in the circulation of the adult subjects.

Insulin binding and degradation by human erythrocytes at physiological temperature.

When evaluated at 15 C, insulin binding to human erythrocytes is similar to that of human adipocytes fibroblasts, monocytes and placental membranes. At 37 C, however, both insulin binding and degradation by human erythrocytes have a unique character. At this temperature, by the end of the first 30 minutes, erythrocyte specific insulin binding is 3 to 4% of the total available insulin. This percentage of binding remains until the end of the first hour, then for the next four hours, increases linearly to 24%. Intact erythrocytes had negligible degradation of the free 125I-insulin but 56% of the 125I-insulin associated with the erythrocytes was degraded after five hours of incubation at 37 C. The degradation of the bound insulin was determined to be an intracellular property of erythrocytes. This degradation may be the mass action driving force responsible for the increased association of 125I-insulin observed after one hour of incubation. On the other hand, erythrocyte ghosts reached a steady state with 2% of the 125I-insulin bound after 1.5 hours of incubation at 37 C. More than 94% of the bound and free insulins were intact after 5 hours of incubation. These observations indicate, for the first time, that erythrocyte insulin degrading activity is localized inside the cells, not in their membranes, and that the human erythrocyte with its insulin receptors may be one of the important cell types in the metabolism of insulin.

Insulin degradation by human erythrocyte lysates.

In vitro hemolysates of isolated human erythrocytes degrade 125I-labeled insulin. Ten- to 100-fold dilutions of the hemolysate give a proportionately decreased degradation of 125I-labeled insulin at 37 degrees C, while dilutions of up to eightfold do not. Like the control, diluted "Buffer G" containing 5 mmol/L Tris and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer alone, more than 500-fold dilutions of the hemolysate or boiled hemolysate (in buffer) caused negligible (less than 1%) degradation of the labeled insulin. We conclude that accurate insulin-binding data during erythrocyte insulin radioreceptor assay under optimum conditions (Clin. Chem. 23: 1590-1595, 1977) depend on avoiding or minimizing hemolysis.