I. Søndergaard

Imipramine: clinical effects and pharmacokinetic variability.

Sixty-six hospitalized depressed patients were treated for 4 weeks with imipramine (Tofranil®) 225 mg/day. Blood samples were drawn twice weekly 15 h after the last drug intake, and IP and DMI concentrations in plasma were assayed by quantitative in situ thin-layer chromatography. Clinical rating was carried out once weekly by Hamiltons Rating Scale (HRS), Becks Depression Inventory, WHO Depression Scale (Quantitative Part), and a side-effect scale. The patients were classified on the basis of the WHO Depression Scale (Qualitative Part) as ‘endogenous’ (N=37) or ‘non-endogenous’ depressions (N=29). Antidepressive effect was evaluated on the basis of the posttreatment rating scores.In patients classified as ‘endogenous’ depressions all 12 responding patients (HRS≦7) had plasma levels of IP>45 μg/l and DMI>75 μg/l, whereas 11 out of 14 nonresponding patients (HRS≧16) had plasma levels of one or both compounds below these limits. Ten out of 12 responders had levels of IP+DMI above 240 μg/l, and all nonresponders had levels of IP+DMI below this limit. Patients with partial response (HRS: 8–15) formed an overlapping group. There was no sign of an upper plasma level limit for the antidepressive effect of imipramine.The plasma level/effect relationship was less clear in patients with ‘non-endogenous’ depressions, since several of them responded at low plasma levels.Some relationship between effect on blood pressure (orthostatic effect) and high plasma levels of IP and DMI was found.Using a plasma level limit of IP≷45 μg/l and DMI≷75 μg/l, it was possible to predict the response of the ‘endogenous’ depression group for 10 out of 12 responders and 10 out of 14 nonresponders on the basis of plasma level measurements obtained after 1 week of treatment.

The Ige and IgG subclass antibody response in patients allergic to yellow jacket venom undergoing different regiments of venom immunotherapy

We developed a three-layer immunoradiometric assay for quantitation of IgG antibodies of all four subclasses to YJV. We studied the IgE and IgG subclass antibody response to YJV in 31 patients allergic to YJV who were undergoing three different kinds of venom immunotherapy. Group A received weekly single injections with alum-adsorbed venom, group B received weekly clustered injections with aqueous venom, and group C received fortnightly clustered injections with aqueous venom during the increasing dose phase of our study. All patients received alum-adsorbed venom during maintenance therapy. Results from the first 6 months of observation are reported. After 6 months of therapy the IgE antibody level rose significantly in group A, was unchanged in group B, and tended to fall in group C. The fall in IgE antibody level in group C correlated significantly to the pretreatment IgE antibody level. The IgG subclass antibody assays measured IgG antibodies of different subclasses in comparable units. No IgG2 or IgG3 antibodies to YJV were found. Before the start of immunotherapy, 23 patients had significant concentrations of IgG1 antibodies to YJV, and 14 had significant concentrations of IgG4 antibodies. In group A the IgG1 antibody level rose significantly after 6 months, and the IgG4 antibody level rose significantly after 3 months. In group B the IgG1 antibody level rose after 2 weeks and the IgG4 antibody level rose after 3 weeks. In group C the IgG1 antibody level rose after 2 weeks and the IgG4 antibody concentration rose after 8 weeks. When the maintenance dose was reached, the IgG1 antibody level in group C was significantly higher than that in group A. The possibility that IgG1 antibodies formed during venom immunotherapy take part in a feedback inhibition of the IgE antibody production is discussed.

Steady-state kinetics of imipramine in patients.

Steady-state plasma level kinetics were studied in 76 patients given imipramine (IP) 150 to 225 mg/day for 2–5 weeks. IP was given in three divided doses at 8.00 a.m., 1.00 p.m. and 5.00 p.m. Plasma concentrations of IP and its active metabolite desipramine (DMI) were determined by quantitative in situ thin-layer chromatography. The plasma levels of IP and DMI showed pronounced flucutations throughout the day with a ratio of about 2 between highest and lowest level. Patients with steady-state levels of IP and/or DMI below 50 μg/l reached this within 1 week of treatment. Patients with higher steady-state levels reached steady-state concentrations within 2–3 weeks. There were some intraindividual fluctuations in plasma levels from week to week after steady state had been reached (coefficient of variation: 10–20%). Interindividually, the steady-state levels corrected to a dose of 3.5 mg/kg per day varied considerably: IP: 6–356 μg/l, DMI: 24–659 μg/l and IP+DMI: 58–809 μg/l. The steady-state plasma levels showed a skew distribution that became normal by logarithmic transformation. The IP/DMI ratio ranged from 0.07 to 5.5 with a median value of 0.47. Compared to data from amitriptyline treated patients the IP/DMI ratios had significantly lower median value and larger variation than the corresponding plasma level ratios of amitriptyline/nortriptyline. Several statistically significant differences in steady-state levels between age groups were found. For IP: Women aged 30–39 had lower levels than women aged 20–29, 40–49, and 50–59, and men aged 50–59 and 60–65; men aged 30–39 had lower levels than men aged 60–65. For DMI: Women aged 30–39 had lower levels than women aged 50–59.

Clustered immunotherapy with Yellow Jacket venom. Evaluation of the influence of time interval on in vivo and in vitro parameters.

To evaluate difference in clinical efficacy, side effects, in vivo and in vitro parameters, 25 patients allergic to Yellow Jacket were treated with clustered immunotherapy using either 7 or 14 days interval between clusters. Twenty‐one patients completed the 6 months’ treatment period and four were withdrawn due to adverse reactions (2 cases of anaphylactic shock). Sixteen patients were challenged by in‐hospital sting and the clinical efficacy was complete. Local side effects were observed in the majority of patients, but only rarely limited the course of immunotherapy. Skin sensitivity estimated as the venom concentration eliciting a wheal equal to histamine HCl 0.1 mg/ml using intradermal test was significantly reduced after 6 months of treatment. Specific IgE showed an initial increase, thereafter declining to pretreatment levels. IgG subclasses were determined by a triple antibody assay. Only subclasses 1 and 4 showed response. Subclass 4 showed a steady increase contrary to subclass 1 which decreased after reaching maintenance dose. No unambiguous relation between either the absolute value or the change of IgG1 and IgG4 at the time of challenge was observed in the patients who tolerated a sting. Furthermore, the IgG response was not correlated to the cumulative dose of venom administered. No simple regulatory function of IgG subclasses in the skin and IgE response was found, and the occurrence of local side effects did not seem to be determined by IgG antibodies. We conclude that clustered immunotherapy with Yellow Jacket venom is highly effective and that the frequency of side effects is acceptable. Neither the IgE nor the IgG response is significantly different with a 7‐ and 14‐day interval between clusters, indicating that a protective dose of venom might be reached within 1 month.

A Three‐Layer Immunoradiometric Assay for Determination of IgG Subclass Antibodies in Human Sera (“IgG Subclass RAST”)

We report the development of a three‐layer immunoradiometric assay (TIRA) for measurement of IgG antibodies of all four subclasses in human sera. The first layer consists of diluted human serum, the second layer is monoclonal mouse antibodies to human IgG subclasses., and the third layer is 125I‐labelled rabbit anti‐mouse IgG. Monoclonal anti‐IgG1. anti‐IgC3 and anti‐IgG4 reacted only with their complementary IgG subclass, whereas the anti‐lgG2 showed slight cross‐reactivity to immunoglobins of other subclasses and classes and to light chain proteins. The observed cross‐reactivity was found to be without importance, when the TIRA was applied to measurement of IgG subclass antibodies. Equipotency was established by use of appropriate dilutions of the monoclonal antibodies, and the assay was calibrated by use of human reference serum. The TIRA therefore permits reliable inter‐individual and intra‐individual comparisons of the IgG antibody response in all four subclasses. Von‐sped fie binding obtained with pooled normal human serum was below 0.33′#. Inter‐assay coefficient of variation was between 18 and 27%, The TIRA was applied to measurement of IgG subclass antibodies to timothy grass pollen in sera from grass pollen allergies undergoing immunotherapy.

Central and peripheral 5-HT uptake in rats treated chronically with femoxetine, paroxetine, and chlorimipramine.

In rats acute treatment with 5-HT uptake inhibitors antagonize p-chloro-amphetamine (PCA)-induced hypermotility and 5-HT depletion from brain. To compare the acute and chronic effect on neuronal 5-HT uptake these two effects of PCA were studied after 4 weeks feeding with diets containing the three 5-HT uptake inhibitors femoxetine, paroxetine, and chlorimipramine. Prolonged treatment with 5-HT uptake inhibitors reduce blood 5-HT due to blocked 5-HT uptake into platelets. Blood 5-HT and plasma concentrations of the 5-HT uptake inhibitors were determined.Femoxetine and paroxetine antagonized both effects of PCA and depleted blood 5-HT at plasma concentrations equivalent to those of patients treated with these substances. The results strongly indicate preservation of inhibited 5-HT uptake into neurons and platelets during prolonged treatment of rats with femoxetine and paroxetine.The plasma concentrations of chlorimipramine at the highest dose were lower than those of chlorimipramine treated patients, but the concentrations of desmethylchlorimipramine (DCIP) were higher. Inhibition of PCA-induced hypermotility and partial blood 5-HT depletion was obtained at these relatively low plasma levels of the substance itself, but the effect of PCA on brain 5-HT was not changed. In rats, it appears difficult under chronic conditions to keep the plasma concentrations of chlorimipramine high enough to inhibit neuronal 5-HT uptake without simultaneous high toxic concentrations of DCIP which is a weak 5-HT uptake inhibitor. The inhibition of PCA-hypermotility by chlorimipramine is probably caused by the dopamine receptor blockade of this substance.

Image processing and pattern recognition algorithms for evaluation of crossed immunoelectrophoretic patterns (Crossed radioimmunoelectrophoresis analysis manager; CREAM)

A computerized method for automatic evaluation and comparison of crossed immunoelectrophoretic and crossed radioimmunoelectrophoretic patterns that requires limited hardware resources has been developed. For the initial reading of the plates an ordinary video camera is used. Feature extractors that allow the computer to recognize a point on the precipitation curve as being a peak point have been developed. After this automatic procedure the program allows for an interactive menu-driven proofreading phase during which it is possible to force the system to take into consideration any number of extra points along the precipitation curve in the curve-fitting process. The system has been tested on crossed immunoelectrophoretic patterns as well as crossed radioimmunoelectrophoretic patterns and it has been shown that the system can recognize the same precipitation curves on different immunoplates and autoradiographs. In addition, the system reports the mean, the variance, and the area of the precipitation curves, thus allowing not only a qualitative comparison of two or more plates but also a quantitation of individual antigens or antibodies.

Quantitative Determination of 1,4‐Methyl‐Imidazoleacetic Acid in Urine by High Performance Liquid Chromatography

A reversed phase ion‐pair high performance liquid chromatographic (HPLC) method for quantitative determination of the major histamine metabolite 1,4‐methyl‐oleacetic acid (1,4‐MIAA) in urine is described. The sample handling is minimal as the sample only has to be centrifuged and diluted before analysis, in contrast to earlier gas chromatographic and thin‐layer chromatographic methods. The method correlates well with a thin‐layer chromatographic determination (r=0.79, n=15), and results from analysis of urine from different patients suffering from mastocytosis, chronic urticaria, atopic dermatitis and asthma are presented together with samples from normal individuals. Most of the patients analysed showed increased excretion of 1,4‐MIAA compared with the controls. Future applications of the method, including analysis on patients with food allergy and patients with abnormalities in their histamine metabolism, are discussed.

Maxisorp.: RAST A sensitive method for detection of antigen-specific human IgE in culture fluids

For determination of allergen‐specific IgE in cell culture supernatants and other highly diluted IgE preparations a radioallergosorbent test (RAST) based on high adsorption polystyrene test tubes has been developed (“Maxisorp RAST”). Cladosporium herbarum extract was used as a model allergen but timothy grass pollen, house dust mite and dog dander showed similar results. The test showed specificities of both allergen and immunoglobulin isotype and significant correlations (r= 0.67–0.88) with established RAST procedures were found. Based on immunosorbent‐purified allergen‐specific IgE the estimated sensitivity was within the order of 150‐300 pg allergen‐specific IgE per ml. The within‐assay variation was 4‐9% and the inter‐assay‐variation 17–29%. The Maxisorp RAST is useful as an inhibition assay for quantitating allergenic activity down to 0.1 biological units/ml of allergen extracts.

A computational approach to the description of individual immune responses. IgE and IgG-subclass allergen-specific antibodies formed during immunotherapy.

Detailed evaluation of the IgE and IgG‐subclass immune response during immunotherapy can now be performed by crossed radio immunoelectrophoresis (CRIE). Some new concepts are introduced facilitating the handling of the vast amount of data obtained by quantitating the immune response. These concepts are “distance” between antibody responses and “immune response width”. The 20 patients included in this study were pollen‐allergic patients who underwent specific immunotherapy in a 3‐year prospective study. It was found that the immune response during immunotherapy was restricted to IgG1 and IgG4 antibodies. The semi‐quantitative CRIE analysis correlated with the RAST analysis for the IgE samples before start of immunotherapy, for the IgG1 samples at week 12, and for all the IgG4 samples. During immunotherapy the number of IgG1 antibodies directed to the different antigens increased towards 11 antigens and decreased towards six. For the IgG4 antibodies the number of reactions increased towards 15 antigens and decreased towards four. The increase is generally paralleled by an increase in the quantitative immune response as well. For some of the antigens a rise in the IgE antibodies is contrasted by a fall in the IgG4 antibody response, and for other antigens the opposite was true, indicating a regulatory mechanism between the IgE and the IgG4 synthesis. The IgE immune response to a number ively diminished during the period of immunotherapy when IgG1 was present early (week 12) in the period, and for other antigens there was a rise in IgE without an early IgG1 antibody response. This suggests that IgG1 can have a regulating influence on the IgE synthesis. Finally, we have found that IgE antibodies with specificities not present in the samples taken before immunotherapy were formed during immunotherapy. These new IgE antibodies do not, however, seem to impair the outcome of treatment.