Rational design of contact guiding, neurotrophic matrices for peripheral nerve regeneration.

Nerve guides filled with magnetically aligned hydrated gels of type I collagen have been shown to impart strong contact guidance cues to elongating neurites in vitro and to increase the number of regenerating axons in vivo relative to an isotropic collagen gel. We have formulated and analyzed a model to determine the conditions under which the target concentration of nerve growth factor (NGF) to support axonal growth can be sustained by entrapping either NGF-secreting cells or NGF-releasing polymer microspheres in the aligned gel. The equation describing NGF concentration with a distributed source term was solved after experimental determination of (1) the rate of NGF release from PLGA 85/15 microspheres, (2) the NGF diffusion coefficient in the gel and nerve guide membrane containing the gel, and (3) the maximum microsphere loading that does not compromise the magnetic alignment of collagen fibrils. We find that for a rat sciatic nerve, when using a 1 mm diameter nerve guide within a commercially available collagen membrane, the microsphere loading limit will prevent the construct's capacity to sustain the target NGF concentration of 1 ng/ml at two months when either wild type Schwann cells or PLGA 85/15 microspheres are used as the NGF source. This target concentration, however, will be maintained when transfected cells described in the literature to hypersecrete NGF are used, or when the microspheres are used if the permeability of the nerve guide membrane can be moderately decreased. For a human median nerve, when using a 5 mm diameter nerve guide within a commercially available membrane, the microspheres are capable of sustaining NGF concentrations above 1 ng/ml to at least 75 days without the need to decrease membrane permeability.

Theoretical analysis of inward hemispheric release above and below drug solubility.

A detailed analysis of inward diffusional drug release from devices with hemispheric and related geometries is presented. When drug is loaded below its solubility, an infinite series describes drug concentration profiles and release kinetics, with an excellent approximation resulting when only one term of this series is retained. A connection between this geometric setting and diffusion in constricted porous domains is pointed out, as is the utility of mean first passage times and mean residence times derived for this model. For the case of drug loaded above its solubility, the pseudosteady state (PSS) approximation of Béchard and McMullen [J. Pharm. Sci. 77 (1988) 222] is compared against numerical results calculated for the full model in which the PSS assumption is removed. A close match is observed. Asymptotic analysis of the PSS expressions shows that the previously used zero-order release assumption is not quite correct, even at later times, and this affects parameter estimation procedures. A comparison between the model of Béchard and McMullen and earlier obtained experimental data [J. Pharm. Sci. 72 (1983) 17] reveals some qualitative discrepancies that are yet to be explained.

Threshold modeling of autonomic control of heart rate variability.

Even in the absence of external perturbation to the human cardiovascular system, measures of cardiac function, such as heart rate, vary with time in normal physiology. The primary source of the variation is constant regulation by a complex control system which modulates cardiac function through the autonomic nervous system. Here, we present methods of characterizing the statistical properties of the underlying processes that result in variations in ECG R-wave event times within the framework of an integrate-and-fire model. We first present techniques for characterizing the noise processes that result in heart rate variability even in the absence of autonomic input. A relationship is derived that relates the spectrum of R-R intervals to the spectrum of the underlying noise process. We then develop a technique for the characterization of the dynamic nature of autonomically related variability resulting from exogenous inputs, such as respiratory-related modulation. A method is presented for the estimation of the transfer function that relates the respiratory-related input to the variations in R-wave event times. The result is a very direct analysis of autonomic control of heart rate variability through noninvasive measures, which provides a method for assessing autonomic function in normal and pathological states.

Age effects on interrelationships between lung volume and heart rate during standing.

To determine the effects of aging and posture on the relationship between respiration and heart rate (HR), we collected 5 min of lung volume and R-R interval data from 7 young (27 +/- 3 yr, mean +/- SD) and 10 old (69 +/- 6 yr) healthy humans during spontaneous breathing while they were supine (SU) and standing (ST). Lung volume and HR power spectra and transfer functions between lung volume and HR were estimated. Age and position effects and age-position interactions were determined by analysis of variance for repeated measures. Older subjects had a lower and more variable respiration rate (P < 0.03, P < 0.04), but both age groups exhibited decreased rate of respiration and increased tidal volume with ST (P < 0.05, P < 0.005). ST decreased lung volume-to-HR transfer function magnitude in both groups (P < 0.07). The more marked age-related differences were in phase angle. Both SU and ST phase angles were greater in older subjects (P < 0.003). ST decreased phase angle in young but increased phase angle in older subjects (P < 0.001). In conclusion, respiration, and respiration-HR interrelationships are altered by aging, with increased time delays between lung volume and HR and altered relationships with ST.

Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization.

We describe a PCR-based method for the generation of plasmid multimers that can be directly transformed into Bacillus subtilis with very high efficiency. This technique is particularly useful for the generation of large libraries of randomly mutagenized genes, which are required for the optimization of enzymes by directed evolution. We subjected the gene coding for the protease subtilisin to six consecutive rounds of PCR at three different levels of mutagenicity. The resulting 18 populations were cloned using our PCR multimerization protocol, and the mutation frequencies were determined by DNA sequencing. The resulting data demonstrate that the mutation frequency during PCR can be controlled by adding varying concentrations of manganese chloride to the reaction mixture. We observed a bias in the type of base pair changes with A and T being mutated much more frequently than C and G. We determined the fraction of active clones in all populations and found that its natural logarithm is proportional to the average mutation frequency of the populations. These data reveal that a fraction of about 0.27 of all possible mutations leads to the inactivation of the subtilisin gene, which provides a measure for its structural plasticity.

Water vapour sorption behaviour of copolymers of N, N-diethylaminoethyl methacrylate and methyl methacrylate.

Copolymers of N, N-diethylaminoethyl methacrylate (DEA) and methyl methacrylate (MMA) were synthesized in ethanolic solution and characterized in terms of reactivity ratios, densities and water vapour sorption. For the reaction conditions studied the copolymerization is essentially random. Polymer densities, determined by centrifugation in a density gradient, range from 1.10 for pDEA to 1.13 for p(DEA/MMA) 52/48 mol%. Flory-Huggins chi parameters were determined by isopiestic water vapour sorption, and were found to depend on both the comonomer ratio and the water content. An exceptionally strong dependence of chi on water content (or equivalently, polymer volume fraction) suggests that Flory-Huggins theory is not the proper theory to apply, especially for the polymers with the highest MMA content. Analysis of the data in terms of the Zimm-Lundberg cluster theory reveals that as the polymer becomes more hydrophobic and glassy with increased MMA, the sorption becomes more nonuniform in the polymer.

Age and autonomic effects on interrelationships between lung volume and heart rate.

To determine effects of aging and autonomic input on interrelationships between respiratory and heart rate variability, we collected 5 min of lung volume of R-R interval data from 7 young [27 +/- 3(SD) yr] and 10 older (69 +/- 6 yr) healthy supine humans before and after double pharmacological autonomic blockade with propranolol (0.2 mg/kg iv) and atropine (0.04 mg/kg iv). Estimates of respiratory and heart rate power spectra and linear transfer functions between the two groups were generated by Fourier analysis. Age, double blockade effects, the age-drug interactions were determined by analysis of variance for repeated measures. Basal R-R intervals were unaffected by age. Double blockade decreased R-R intervals and variability in both age groups (P < 0.0001), but R-R intervals decreased less in older than in young subjects (P < 0.0001). In contrast, basal respiratory intervals and standard deviation were greater in older subjects (P = 0.05) and were unaffected by double blockade in young and older subjects. Lung volume-to-heart rate spectral coherence was highest at frequencies associated with respiration and greater in young than in older subjects (P < 0.07). Double blockade decreased lung volume-to-heart rate variability transfer function magnitude (P < 0.007) and increased phase angle (P < 0.02) without age effects or age-drug interactions. In conclusion, heart rate, respiration, and respiration-heart rate interrelations are altered by aging, and double autonomic pharmacological blockade does not eliminate all age-related differences.

pH, salt, and buffer dependent swelling in ionizable copolymer gels: tests of the ideal Donnan equilibrium theory.

We present results of equilibrium swelling studies of the ionizable copolymer gel, methyl methacrylate/N,N-dimethylaminoethyl methacrylate 70/30 mol%, in buffered and unbuffered electrolyte solutions. The experimental conditions were designed to demonstrate the sensitivity of swelling in ionized gels to the electrolyte composition of the external solution. In general, gel swelling as a function of solution ionic strength is shown to be highly nonmonotonic and is particularly sensitive to the valence and concentrations of ions present in solution. A rigorous test of ideal Donnan equilibrium theory shows that the latter is unable to explain all the data in a self-consistent manner. However, a heuristic procedure based on the ideal Donnan theory can predict qualitatively the observed trends. While not quantitative, this heuristic approach provides considerable insight into the mechanisms underlying the swelling behavior under various solution conditions. Possible causes of nonideal behavior are discussed, and some observed specific ion effects are reported and discussed.

Analysis of enzyme specificity by multiple substrate kinetics.

Multiple approaches for screening large sets of compounds for a specific function are of growing interest. The use of substrate mixtures to characterize the specificity of enzymes has been limited so far to compounds with similar kinetic parameters, because the data were analyzed by applying the kinetics of two competing substrates. In this study we introduce a statistical method for the analysis of reactions with many competing substrates which makes use of the specific features of multiple substrate kinetics. It is assumed that the relative concentrations of all substrates in a mixture can be monitored by high-performance liquid chromatography or a similar technique. Relative second-order rate constants, i.e., kcat/KM values, can be calculated for all substrates in the mixture from the resulting data set. The calculation uses the fact that there is a relationship between the concentrations of all pairs of substrates in the mixture. As a result, the precision of the calculated parameters is increased and the range of kinetic constants that can be obtained from one experiment is considerably expanded. Simulations demonstrate that the precision in the kinetic parameters increases with the number of substrates in the mixture. In fact, estimation of ratios of rate constants can be improved (or made possible) for substrates with order of magnitude differences in reactivity by adding "dummy" substrates with intermediate reactivities, even though the rate constants for dummy substrates are themselves of no intrinsic interest.

Buffer effects on swelling kinetics in polybasic gels.

The swelling kinetics of polybasic gels consisting of copolymers of methyl methacrylate and dimethylaminoethyl methacrylate are studied in solutions at various acidic pH values, with monoacidic derivatives of acetic acid added as buffers. The effects of solution pH, as well as buffer pKa and concentration, on swelling rate are assessed. Gel swelling rate shows a nearly linear dependence on the concentration of nonionized buffer in the solution, as determined by the Henderson-Hasselbach equation. This result is explained in terms of the increased availability of protons that are carried by the nonionized buffer to bare amines on the gel. In fact, the so-called pH sensitivity of these gels, under these conditions, can be attributed mainly to the effect of pH on the nonionized buffer concentration. A practical consequence is that these gels may not reliably mediate pH-sensitive swelling-controlled release in oral applications, since the levels of buffer acids in the stomach (where swelling and release are expected to occur) generally cannot be controlled. However, the gels may be useful as mediators of pH-triggered release when precise rate control is of secondary importance.

Long-term structural changes in pH-sensitive hydrogels.

The long-term swelling properties of lightly cross-linked copolymer hydrogels consisting of methyl methacrylate (MMA) and N,N-dimethylaminoethyl methacrylamide (DMAA) were studied as a function of pH at 25 degrees C and 37 degrees C. In acidic pH regions, the swelling equilibria were found to be stable over 200 d. In alkaline pH environments, however, the 'equilibrium' swelling increases slowly with time. Gas chromatography of the supernatant shows that substantial methanol is produced, along with trace amounts of N,N-dimethylethylene diamine. Thus, the primary mechanism underlying the structural changes appears to be hydrolysis of ester groups in the MMA side-chains, with a much smaller contribution due to amidolysis of the DMAA side-chains. The implications of these structural changes for the application of this hydrogel, as well as other related hydrogels as long-term implantable biomaterials, are discussed.

CV 205-502 treatment of macroprolactinomas.

CV 205-502, a benzoquinoline, is a new nonergot dopamine agonist compound which has been shown to be effective in lowering PRL levels in normal volunteers and in hyperprolactinemic women. Seven patients (4 men and 3 women) presenting with hyperprolactinemia due to macroprolactinoma were treated with CV 205-502 given as a single daily dose at bedtime for up to 12 months. Six patients presented with impaired gonadal function and 2 with galactorrhea. All patients but one had previously been treated with bromocriptine and 4 had undergone pituitary surgery (3 with complementary radiotherapy). Six patients responded within a few weeks to CV 205-502 treatment, PRL levels being normalized (4 patients, 0.075 to 0.150 mg/day) or significantly reduced to restore normal gonadal function (2 patients, 0.225 mg/day). The seventh patient, who had previously been resistant to bromocriptine, also failed to respond to CV 205-502 treatment even after high doses (0.450 mg/day). Under CV 205-502 treatment, follow-up with magnetic resonance imaging revealed a reduction in tumor size of up to 52% of the initial volume in the "PRL-responders" whereas an increase in tumor size was observed in the "nonresponding" patient. No biological disturbance appeared during CV 205-502 treatment and the drug tolerance was very good, with mild side-effects being reported by only 2 patients. In conclusion, CV 205-502, given once daily, appears to be a safe and effective alternative to other dopamine agonists in the treatment of macroprolactinoma, by reducing hyperprolactinemia and tumor size. It was, however, of no benefit in the one patient whose macroprolactinoma had been resistant to bromocriptine.

Comparison and critique of two models for regional drug delivery.

A simple stochastic recirculatory formalism is used to compare models of regional drug delivery due to Hunt et al. and Boddy and Aarons. It is shown that these two models are equivalent when regional delivery is ideal. The latter model has the advantage of simplicity. However, the former model appears more useful in relating predictions to experimentally accessible quantities. Neither model is sufficiently general to cover all possible topologies of regions associated with drug response and toxicity. Knowledge of this topology is essential in determining the drug targeting index. The underlying assumptions of the models are discussed, and situations where these assumptions may break down are identified. Finally, it is noted that the analysis of regional delivery may also apply to metabolite and prodrug kinetics.

Binaural processing of noisy stimuli: internal/external noise ratios for diotic and dichotic stimuli.

A set of ten digitized statistically similar Gaussian maskers was used in one-internal tone-in-noise detection experiments under diotic (NoSo) and dichotic (NoS pi) interaural conditions. Stimulus/response matrices were generated for each masker in the presence or absence of a target 500-Hz tone. For both NoSo and NoS pi, nonparametric analyses show that response probabilities and sensitivities vary significantly across noise waveforms, indicating a considerable external noise component in subject response variability. A parametric model is developed that maps individual stimulus waveforms onto a decision axis, facilitating evaluation of internal/external noise variance ratios. For both NoSo and NoS pi, internal and external noise variance are of similar magnitude.

Effects of the novel opiate antagonist, SDZ 210-096, on luteinizing hormone secretion in the rat.

SDZ 210-096 is a novel 14 beta-benzyl morphinan derivative with antagonistic actions at mu and kappa opiate receptors. In the present experiments this compound has been characterized for luteinizing hormone (LH) secretion-stimulating effects in a variety of experimental paradigms in the rat. In juvenile female rats, serum LH levels were elevated markedly after s.c. or p.o. administration of SDZ 210-096; ED50 was less than 1 mg/kg p.o. at 60 min. Similar effects were seen in the male rat, with p.o. ED50 values of 1.5 and 2.8 mg/kg at 60 and 120 min, respectively. Serum LH levels were likewise elevated by this compound in adult female rats (both proestrous and diestrous). SDZ 210-096 maintained its LH secretion-stimulating capability in both male and female rats after up to 10 days of administration. Its effects were inhibited in a competitive manner by a mu (SDZ 202-250) and a kappa (bremazocine) opiate agonist. Moderate effects on prolactin and adrenocorticotropic hormone secretion (inhibition and stimulation, respectively) were observed. SDZ 210-096 exhibited specific high affinity binding to brain opiate binding sites. These results demonstrate that in the rat SDZ 210-096 is a potent, p.o.-active LH secretion stimulator, effective after repeated as well as acute application.

A comparison of personality characteristics, family relationships, and drug-taking behavior in low and high socioeconomic status adolescents who are drug abusers.

Low socioeconomic status (SES) and high SES adolescents who met the DSM-III criteria for drug abuse were compared with respect to personality characteristics, family relationships, and drug-taking behavior. SES was determined by parents' income, education, and occupation. The two groups did not differ significantly with respect to self-esteem, quality of family relationships, impulse control, moral development, and drug-taking behavior. However, high SES adolescents were more depressed (p less than .009) and more anxious (p less than .02) than low SES adolescents, and these findings lent support to the argument that low and high SES drug abusers differ in personality and therefore require different kinds of treatment interventions.

Commentary to "Linear and Nonlinear System Approaches in Pharmacokinetics. How much do they have to offer? I. General considerations".

The review by Veng-Pedersen will hopefully stimulate workers in the field to consider systems techniques more thoroughly. As these techniques are well developed in the engineering fields, students should be encouraged to take relevant courses. In this reviewer's opinion, understanding systems theory will allow pharmacokineticists to "cut through the fat" in the derivation of relationships between kinetic phenomena. However, it should be recognized that the system approaches described in Veng-Pedersen's review probably do not by themselves solve the major problems of response approximation and prediction.

Hypothalamo-hypophyseal-gonadal function in the rat following administration of the novel and selective D-1 agonist CY 208-243.

The effects of the novel and selective dopamine D-1 agonist CY 208-243 on the rat hypothalamo-hypophyseal-gonadal (HHG) axis were studied. CY 208-243 did not modify the concentration of luteinizing hormone (LH) in serum from female or male rats, and had no effect upon opiate antagonist-induced stimulation of LH secretion in male rats. CY 208-243 did not inhibit ovulation in cycling female rats. Thus, D-1 receptor activation by systemic drug administration does not alter HHG function in rats.