Regulatory science: a special update from the United States Food and Drug Administration: Preclinical issues and status of investigation of botanical drug products in the United States.

A recent survey was conducted across the therapeutic divisions within the CDER, U.S. FDA regarding the number of submissions related to botanical drug products over the past ten years. The overall number of botanical submissions as expressed in the parenthesis are as follows: 1990 (1), 1991 (4), 1992 (4), 1993 (5), 1994 (6), 1995 (5), 1996 (13), 1997 (16), 1998 (10). In the total of 64 counted, 50 of them are submitted in original IND and the rest (14) in pre-IND format. The therapeutic categories are focused on dermatological and topical (19), anti AIDS/antiviral (12), oncologic (13), neuropharmacologic (8), endocrine and metabolic (3), urologic (2), tobacco (2), and cardio-renal products (1). The regulatory actions taken on these submissions showed that 68% of them are evaluated as safe to proceed for the human trials, while the rest (32%) of submissions required agency's regulatory guidance. Among the submissions that required further guidance, 81% were deficient in preclinical pharmacology/toxicology information and the rest (19%) lacks information in other areas (chemistry, clinical protocols). Following agency's guidance, 93% of the submissions that were put on hold were allowed to proceed. In summary, a total of 94% of all the botanical INDs submitted to the agency were allowed to proceed without additional animal toxicity studies conducted. In conclusion, this survey indicates that the growing public interest in botanical supplements has prompted more formal evaluation of the efficacy/safety claims of these products.

On the prebiotic role of iron and sulfur.

Thermodynamic data (Sillen, L. G.: 1966, Arkiv Kemi, 25, 159) indicate that there is little support for the idea that metabolic sulfur cycles were involved in prebiotic and early life processes, since under reducing conditions the 'equilibrium' concentration of sulfur in the primordial seas should have been very low, < 10(-8) M. However, it is suggested that metabolic sulfur cycles may have become important when oxygen evolved, when iron(II) ions disappeared from the seas, and when large amounts of sulfur were released from their iron sulfide sources.

Defluoridation of drinking water with pottery: effect of firing temperature.

Excessive fluoride (F) in drinking water should be removed, but simple, inexpensive methods of fluoride removal are not readily available. This study examines the F(-)-binding capacity of clay and clayware, especially the effect of the firing temperature on the F(-)-binding process. A series of pots were made from ordinary potter's clay and fired at 500-1000 degrees C. Likewise, small clay bricks were fired and then crushed and sieved. NaF solutions containing 10 mg/l F- (10 ppm F-) were prepared. Suitable aliquots of the solutions were poured into clay pots or exposed to powdered clayware. Samples were taken at storage periods of 30 min to 20 days and analyzed for F- by ion-selective electrodes. The rate and capacity of F(-)-binding in the clayware varied with the firing temperature. Clay fired at approximately 600 degrees C was most effective. Temperatures over 700 degrees C caused a decline in F(-)-binding, and pottery fired at 900 degrees C and above seemed unable to remove F- from water. Pots fired at 500 degrees C or less cracked in water. The findings indicate that clayware, fired at an optimal temperature, may be of practical value for partial defluoridation of drinking water.

Small-angle scattering study of alpha 1 inhibitor III from rat blood plasma.

The alpha 1 proteinase inhibitor III from rat blood plasma, homologous to the alpha 2-macroglobulin family of proteins, has been studied in solution using small-angle scattering of X-rays and of neutrons: the radius of gyration, Rg, was found to be 4.5 nm, and the largest distance within the molecule, Dmax = 14 nm. When the inhibitor reacts with chymotrypsin or methylamine, the resulting derivatives yield slightly higher Rg-values, 4.7 and 4.85 nm, respectively. The data of the native protein are consistent with a model, the projection of which resembles the letter V and which is formed by the two identical halves of an elliptic cylinder with semi-axes of 2.1 and 5.5 nm and a length of 11 nm. This elliptic cylinder model also explained the scattering from the monomeric complement proteins C3 and C4, as well as that from the monomers of the dimeric and tetrameric alpha 2-macroglobulin family of proteins (Osterberg, R., et al. (1991), Biochemistry 30, 7873-7878). Due to the conformational change occurring when the thiol ester bond is split, the cleft in the V-form seems to be closed; and as a result, the models of the chymotrypsin and methylamine derivatives are more compact than that of the native protein.

The growth of fractal humic acids: cluster correlation and gel formation.

The growth of humic acids, prepared by a gentle method from two different kinds of soils (I and II), has been studied using small-angle neutron scattering at an acidity corresponding to pH 5.0 and 0.10 M ionic strength (NaCl). Humic acids aggregate either to large clusters with a fractal dimension of 2.3 and an average diameter of 1720 A (I) or to clusters with an average diameter of 700 A (II). After storage for 2 days at 4 degrees C, the latter aggregates (II) formed a gel. In a step toward gelation, we observed cluster-cluster interaction from the neutron-scattering data in the form of a correlation peak. These differences in size can be explained by assuming that the smaller particles (II) are trapped into a nonequilibrium state characterized by the temperature-solvent condition. The importance of a humic acid gel network for the conservation of water and nutrients in the environment is discussed.

Correlation between the human and porcine complement system: a small-angle scattering study of cross immunity and methylamine-induced conformational changes of porcine C3 and C4 proteins.

The porcine complement proteins C3 and C4 have been isolated and then characterized using small-angle scattering methods. Within the limits of experimental errors, the porcine proteins are virtually identical with the corresponding human proteins as measured in terms of mol. wt, Mr and radius of gyration, R,: Mr(C3) = 198,000, Mr(C4) = 207,000, and R(C3) = 4.4 nm, R(C4) = 4.5 nm. The C3 and C4 proteins from pigs show cross-immunity with monoclonal antibodies (mAbs) raised against human C3 and C4, respectively. Using the Fab fragments of these mAbs as markers, it is indicated that porcine C3 and C4 undergo a conformational change after reaction with methylamine. The relatively large increase in the radius of gyration observed, delta R = 1.0-1.2 nm, going from the Fab complexes to the Fab complexes of the methylamine derivatives, is similar to that observed for human C3 under similar conditions. This may indicate that methylamine cleaves a labile thiol ester bond supposed to be present within the porcine proteins and that the epitopes interacting with the Fab fragments are very similar to those of the human proteins. Porcine C3 also resembles the human analogue by forming dimers after being subjected to methylamine and dilute lauryl sulphate: Mr = 404,000 and R = 7.9 nm.

X-ray scattering study of hagfish protease inhibitor, a protein structurally related to complement and alpha 2-macroglobulin.

A protease inhibitor from hagfish blood plasma, homologous to human alpha 2-macroglobulin, has been studied in solution using small-angle X-ray scattering; the radius of gyration, R, was found to be 7.0 nm, the molecular weight 340,000 +/- 20,000, and the largest distance within the molecule, Dmax, 22 nm. When the inhibitor reacts with chymotrypsin, its 1:1 chymotrypsin complex is found to be more compact than the native molecule, R = 6.1 nm. A very similar conformational change is observed after the protein is reacted with methylamine. The data are consistent with models consisting of two equal elliptic cylinders with the same size as the one used as a model for the complement proteins C3 and C4 [cf. Osterberg et al. (1989) Eur. J. Biochem. 183, 507-511]. In the model for the native protein, these cylinders are arranged in an extended form, and in the one for the methylamine derivative (or chymotrypsin complex), they are closer together so that the projection of their elliptic surfaces forms an angle of about 70 degrees. These models for the hagfish protease inhibitor were expanded to models for the twice as large human alpha 2-macroglobulin using symmetry operations, and the resulting alpha 2-macroglobulin models were found to agree with those emerged from earlier studies involving electron microscopy and X-ray scattering methods.

Large intramolecular movement in human complement protein C3 induced by methylamine. A small-angle scattering study using monoclonal antibodies as markers.

The reaction of methylamine with complement protein C3, which involves cleavage of a labile thiol ester bond, yields a large intramolecular rearrangement. This is shown by small-angle neutron and X-ray scattering using a Fab antibody as a marker. For the C3(Fab) 1:1 complex, the methylamine reaction yields an increase in the radius of gyration, R, from 4.6 nm to 6.0 nm. In the absence of Fab the corresponding R values increase from 4.4 nm to 5.1 nm. It is estimated that the methylamine-induced increase in R may correspond to a movement of the epitope to a position 5 nm away from the centre of gravity of the C3 molecule. In agreement with this finding, the maximum distance within the C3(Fab) complex increases from 16 nm to 22 nm as a result of the methylamine reaction. In order to explain this conformational change, it is tentatively suggested that the methylamine-induced cleavage of the C3 thiol ester bond leads to a domain rotation within the C3 molecule. In agreement with this idea, the data is consistent with a model which enables a globular domain within the molecule to rotate without redistributing the molecular mass more than that corresponding to the radii of gyration observed.

A neutron scattering study of the ternary complex EF-Tu.GTP-valyl-tRNAVal1A.

The complex formation between elongation factor Tu (EF-Tu), GTP, and valyl-tRNAVal1A has been investigated in a hepes buffer of "pH" 7.4 and 0.2 M ionic strength using the small-angle neutron scattering method at concentrations of D2O where EF-Tu (42% D2O) and tRNA (71% D2O) are successively matched by the solvents. The results indicate that EF-Tu undergoes a conformational change and contracts as a result of the complex formation, since the radius of gyration decreases by 15% from 2.82 to 2.39 nm. tRNAVal1A, on the other hand, seems to mainly retain its conformation within the complex, since the radii of gyration for the free (after correction for interparticular scattering) and complexed form are essentially the same, 2.38 and 2.47 nm, respectively.

Dimerization of human complement proteins C3 and C4 in dilute lauryl sulfate buffer after reaction with methylamine.

In the presence of methylamine and dilute lauryl sulfate (pH 8.0), the human C3 and C4 complement proteins dimerize almost completely. Under these conditions, the related complement protein C5 does not show any tendency to form dimers. This is shown by x-ray and neutron scattering at 9 degrees C and 0.15 M ionic strength. The radii of gyration of the C3 and C4 dimers are very similar, 7.7 and 7.4 nm, and the cross-sectional radii of gyration are the same, 3.4 nm. The scattering curves of the C3 and C4 dimers as well as their Fourier transforms, the p(r)-curves, can be explained by scattering from a model consisting of an elongated elliptic cylinder with semiaxes 6.5 and 2.1 nm and length of 23 nm. This elongated elliptic cylinder model is consistent with the elliptic cylinder model of C4 (Osterberg, R., Eggertsen, G., Lundwall, A., and Sjöquist, J. (1984) Int. J. Biol. Macromol. 6, 195-198) provided that the protein molecules dimerize via their cross-sectional surfaces. Also, the model is consistent with the model of the related protein, alpha 2-macroglobulin, where the four subunits are supposed to form pairwise dimers of an elliptic cylindrical form (Osterberg, R., and Malmensten, B. (1984) Eur. J. Biochem. 143, 541-544).

The condensation of DNA by chromium (III) ions.

Cr(III), one of the most potent inorganic carcinogens, induces condensation of DNA into a very compact product at 37 degrees, as shown by electron microscopy. The condensation begins with the appearing of some supercoil structures and complete condensation occurs at relatively low Cr(III) concentrations; for 3 and 30 mM ionic strength they are 4.5 and 45 microM, respectively. Under these conditions, Cr(III) inhibits the interaction between ethidium and DNA as shown by absorption and fluorescence spectra.

Methylamine-induced conformational change of alpha 2-macroglobulin and its zinc (II) binding capacity. An X-ray scattering study.

Methylamine induces a conformational change of alpha 2-macroglobulin which is very similar to that obtained by proteinase reaction and binding. This was shown by small-angle X-ray scattering at 21 degrees C in 0.03 M Hepes buffer of pH 8.0 containing 0.15 M NaCl and 0.3 mM EDTA. When alpha 2-macroglobulin reacts with methylamine the side maximum virtually disappears from the X-ray scattering curve and the radius of gyration decreases from 7.8 nm to 7.2 nm. The X-ray data of alpha 2-macroglobulin are consistent with an open shape model similar to that deduced via electron micrographs [Schramm, H. J. and Schramm, W. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 803-812]; one projection of the model resembles the letter H; the four subunits are mainly represented as elliptical cylinders which are connected via a central, quite flat cylinder. Zinc(II) ions cause aggregation of alpha 2-macroglobulin even at such a low total zinc concentration as 12.5 microM; for 25 microM zinc(II) concentration, the average molecular mass indicates that the aggregation goes beyond the dimeric stage. Monomeric species of alpha 2-macroglobulin appear to have the capacity specifically to bind 8.0 zinc(II) ions per molecule, which corresponds to two zinc(II) ions per subunit.

Structure of alpha 2-macroglobulin in solution and its interaction with proteases: an X-ray scattering study using the contrast variation method.

X-Ray scattering study of alpha 2-macroglobulin in solvents of variable electron densities (sucrose in water) shows that alpha 2-macroglobulin obeys the invariant volume hypothesis; thus, the structure of the particle is independent of the sucrose concentration of the solution. The particle structure is quantitatively described by a set of parameters such as the gyration radius, R = 8.0 nm, the volume, V = 1200 nm3, and the maximum distance within the particle, Dmax = 25 nm. The contrast dependence of the gyration radius indicates that in alpha 2-macroglobulin the regions of higher electron density are located closer to the center (core) than the regions of lower electron density. The core, which may be the place occupied by the carbohydrate, has a maximum dimension of 16 nm and it can be described as a flat cylinder. X-Ray scattering titrations indicate that alpha 2-macroglobulin forms a 1:2 complex as the main product with both trypsin and chymotrypsin simultaneously as the particle contracts. The formation of a ternary 1:1:1 complex with trypsin and chymotrypsin and the absence of higher complexes indicate that the sites for these proteases are closely related. This is further substantiated by the p(r) functions which are virtually identical for the 1:1:1 and 1:2 complexes.

Small-angle X-ray scattering study of the interaction between human alpha 2-macroglobulin and trypsin.

The interaction between human alpha 2-macroglobulin and trypsin yields a complex of two trypsin molecules and one alpha 2-macroglobulin molecule as the main product. This is indicated from small-angle X-ray titration measurements in 0.02 M Tris/HCl buffer of pH 7.40 containing 0.2 M NaCl and 5 mM EDTA. The radius of gyration for the complex was determined to be 7.1 nm. The complex formation coincides with a decrease in the first side maximum of the scattering curve for alpha 2-macroglobulin. A comparison of the experimental data with those calculated from various models indicates that the data are consistent with the scattering from a hollow cylinder containing two sites, each of which is partly filled with one trypsin molecule.

Ocular irritancy responses to various pHs of acids and bases with and without irrigation.

Acids and alkalies were instilled into the eyes of 2 groups of rabbits; the eyes of one group were washed with tap water 30 s after exposure. Damage seen in washed and unwashed eyes was not always related to pH. Some strong acids with greater acidity than pH 2.5 produced opacities while 0.3% hydrochloric acid with a pH of 1.28 produced no ocular damage. Phenol (5%) and acetic acid (5%) with pHs greater than 2.5 produced damage equivalent to or greater than that produced by equal concentrations (w/v) of the mineral acids. All alkalies with pHs ranging from 11.5 to 13.5 produced opacities and other ocular damage of different degrees depending upon the alkali and its concentration. For example, low concentrations of some alkalies in the pH range from 11.3 to 12.8 produced no ocular changes. The duration of the corneal opacities produced by phenol, 1% sodium hydroxide, acetic acid and anhydrous sodium carbonate and the onset of corneal opacity produced by 5% sulfuric acid, the weak acids and 1% sodium hydroxide were reduced as a result of washing the test eyes 30 s after instillation of the test material. These data suggest that acidity and alkalinity of the test material are not the only factors to be considered in relation to a substances' capacity to produce severe ocular injury. The concentration of the test chemical and its period of contact with the eye prior to washing are also important.

A small-angle X-ray scattering study of the complex formation between elongation factor Tu . GTP and valyl-tRNA Val I from Escherichia coli.

The complex formation between elongation factor Tu (EF-Tu) . GTP and valyl-tRNA Val 1 has been investigated using the small-angle X-ray scattering titration technique. The main species observed is a 1:1 complex with a stability constant log K greater than or equal to 6. The corresponding interaction between EF-Tu . GTP and non-aminoacylated tRNA appears to be much weaker with an estimated log K approximately equal to 4. The radius of gyration determined for the EF-Tu . GTP -- valyl-tRNA Val 1 complex is larger (R = 3.6 nm) than that of EF-Tu . GTP (R = 2.5 nm). Likewise, the maximum distance within this complex is larger (Dmax = 12.5 nm) than the one within EF-Tu . GTP (Dmax = 8.5 nm). These data as well as the p(r) curve are consistent with a multiellipsoid model for the complex. From this model it is indicated that the acceptor stem of tRNA is attached to EF-Tu and that the anticodon stem and loop protrude into the solution.