Exploring the relationship of drug-induced neutrophil immaturity & haematological toxicity to drug chemistry using quantitative structure-activity models.

An investigation of the relationships between physicochemical features of ten antipsychotic drugs and previously reported influence of these drugs on neutrophil maturity was made. A quantitative structure-activity relations (QSAR) approach was adopted, in which several numerical parameters describing physicochemical characteristics of the antipsychotics were estimated. Possible connections between these parameters and neutrophil maturity were explored. Influence of drug physicochemistry on the incidence of agranulocytosis and neutropenia reported in the literature was documented. Overall it was found that drugs with the greatest tendency to induce neutrophil immaturity (chlorpromazine, clozapine and olanzapine) also showed the greatest tendency to cause agranulocytosis and neutropenia. Moreover marked induction of neutrophil immaturity occurred with compounds of moderately amphipathic character, whose amphipathic indices (AI) fell in the range 3-5; higher or lower AI values correlated with less immaturity. Consideration of the QSAR findings suggest that toxicity could be associated with selective uptake into the most fluid intracellular membranes, those of the endoplasmic reticulum and the outer mitochondrial membrane. The AI hazard zone (AI = 3-5) does constitute a predictive tool to assess risk of agranulocytosis and neutropenia arising from antipsychotic and other psychoactive drugs - and not only risk arising from medication but also from experimental or even proposed compounds.

Formation of immature neutrophil leucocytes in schizophrenic patients treated with various antipsychotic drugs: comparisons and predictions.

The neutrophils of schizophrenic patients taking antipsychotic drugs were evaluated. Neutrophil immaturity was assessed by determining mean nuclear lobe number in peripheral blood smears of patients and controls. Subjects were patients medicated with typical (upenthixol (n 6), uphenazine (n 7), haloperidol (n 23), thioridazine (n 15), and triuoperazine (n 6)) or atypical (olanzapine (n 15), risperidone (n 10), and sulpiride (n 7)) antipsychotic drugs. Controls (n 58) were healthy, non-medicated clinical and academic staff. Mean lobe number was determined using light microscopy and examining 300 neutrophils per individual. For subject and control groups, means and medians of mean lobe numbers, and also mean white cell and neutrophil counts, were determined. Means for each group were compared using the Mann-Whitney U test; variances using F ratios. Mean lobe numbers of all patients were decreased compared to controls. The left shift occurring in patients medicated with haloperidol, olanzapine, risperidone, thioridazine, and triuoperazine was signícantat P 0.0001; for upenthixol P 0.001, and for sulpiride P 0.05. The left shift for uphenazine was not statistically signíant. For one patient, mean lobe numbers were obtained before and after medication with olanzapine commenced, and a lowering of mean lobe number was seen. Although the coefficient of variation in the patient groups was large compared to the controls, nevertheless more than half of the patients had mean lobe numbers outside the observed range of values seen in the control population. White blood cell and neutrophil counts in patients and controls were not signiécantly different. This study demonstrated that patients taking antipsychotic drugs have immature neutrophils, but normal total white cell and neutrophil numbers. The effect was seen with both typical and atypical antipsychotic drugs, and is probably drug-induced. It is possible that mean lobe number may predict patients at risk from neutropenia or agranulocytosis, as is also suggested by an analysis of the relative numbers of literature reports of neutrophil pathology for these drugs. It is of interest that olanzapine, which has been considered a haematologically non-hazardous drug, was shown to be associated with a significant decrease in mean lobe number.

Roadmap to approval: use of an automated sterility test method as a lot release test for Carticel, autologous cultured chondrocytes.

BACKGROUND: In February 2004, FDA approved a supplement to our biologics license for Carticel, autologous cultured chondrocytes, to use the BacT/ALERT microbial detection system as an alternative to the compendial sterility test for lot release. This article provides a roadmap to our approval process. The approval represents more than 4 years of development and validation studies comparing the Steritest compact system to the BacT/ALERT microbial detection system. METHODS: For this study, freshly cultured chondrocytes were prepared from a characterized cell bank. Microbial isolates were prepared from either American Type Culture Collection (ATCC) strains or from in-house contaminants. For each test condition, a suspension of chondrocyte cells and test organisms was inoculated into both aerobic media (SA standard adult culture bottles, FA FAN, tryptic soy broth) and anaerobic media (SN standard adult culture bottles, FN FAN, fluid thioglycollate media) and tested for sterility using the Steritest compact system (Millipore, Bedford, MA, USA) and the BacT/ALERT microbial detection system (bioMerieux, Durham, NC, USA). Negative control bottles were inoculated with chondrocytes and no microorganisms. All bottles were incubated for 14 days and read daily. Bacterial growth was determined by either visual examination of Steritest canisters or detection of a positive by the BacT/ALERT system. A gram stain and streak plate were used to confirm positive bottles and negative bottles after 14 days. RESULTS: The detection of a positive by either the Steritest compact system or the BacT/ALERT system was summarized for each organism in each validation study. Data generated from studies reducing the incubation temperature from 35 degrees C to 32 degrees C improved detection times in the automated method compared with the compendial method. Other improvements included the use of FAN aerobic and anaerobic media to absorb the gentamicin contained in the culture media of prepared chondrocyte samples. Chondrocytes alone did not generate positive results in either the compendial method or the automated method. DISCUSSION: Data from validation studies support the use of the BacT/ALERT microbial detection system as an alternative sterility test for Carticel.

Absence of macrocytic anaemia in Alzheimer's disease.

There is an association between Alzheimer's disease (AD) and low serum levels of vitamin B12 and folic acid. Patients also have elevated serum levels of homocysteine and disease progression might therefore be associated with the development of a macrocytic anaemia. We investigated the relationship between disease duration, homocysteine and haematological indices in patients with clinically diagnosed AD and healthy elderly controls. Haemoglobin and platelet counts fell only slightly with increasing dementia duration, but there were no other changes in haematological indices. In particular, macrocytosis and red cell distribution width were unrelated to disease duration and no patients were anaemic. Our results support previous observations that the neurological and haematological features of B12 and folate deficiency are often unrelated in these patients.

Antipsychotic drugs result in the formation of immature neutrophil leucocytes in schizophrenic patients.

Subclinical abnormality of neutrophil populations of patients suffering from schizophrenia and medicated with antipsychotic drugs was evaluated using cellular immaturity as a criterion. Neutrophil maturity of patients and controls was compared by determining mean nuclear lobularity in peripheral blood smears. White blood cell and neutrophil counts were made. Subjects were patients medicated with chlorpromazine (n = 17) or clozapine (n = 48). Controls (n = 58) were healthy, non-medicated clinical and academic staff. Determination of mean lobe number involved assessment of 300 neutrophils per individual. For subject and control groups, means and medians of mean lobe numbers and mean white cell and neutrophil counts were determined. Means for each group were compared using the Mann-Whitney U-test; variances using F ratios. Means of lobe numbers of both patient populations were significantly different (p < 0.0001) compared to controls. Two-thirds of patients had mean lobe numbers outside the control range. Dose-response (mean lobe number) plots were significant for patients medicated with both chlorpromazine and clozapine. White cell and neutrophil counts in patients and controls did not differ significantly. For six patients, mean lobe numbers were obtained before and after medication commenced and all showed lowering of mean lobe number. The mean lobe number of the one patient who subsequently suffered from agranulocytosis was at the low end of the patient range. Thus, patients medicated with antipsychotic drugs typically have immature neutrophils, but normal white cell and neutrophil numbers. This effect is probably drug-induced. Mean lobe number may predict patients at risk from agranulocytosis.

DNA microarray analysis of differential gene expression of 6-year-old rat neural striatal progenitor cells during early differentiation.

EGF-responsive striatal progenitor cells from rat brain have been maintained in culture in the form of neurospheres for six years without exhausting their renewal capacity. The events surrounding differentiation of stem cells in the brain after a long progenitorship remain a mystery. Using DNA microarray analysis we investigated differential gene expression, comparing progenitor cells in their neurosphere state with the cells 24 hours after induction of differentiation. Eighty-one genes showed increased expression in the differentiated condition. Genes associated with cellular growth, neurite outgrowth, and synaptogenesis were activated, including both anti-apoptotic and pro-apoptotic genes. Two transmitter- related genes, acetylcholine receptor-beta and glutamate receptor-beta-unit were also elevated-, these genes not only fit the profile of early neural development, but also reflect the characteristics of striatal neurons. In addition, there are approximately 30 expressed sequence tags (ES7) increased during neural differentiation. Forty-seven genes showed decreased expression; half of them are known genes related to the cell cycle, cell adhesion, transcription, and signaling. Tbe signaling and cell cycle genes may be responsible for the life-long self-renewal. These data demonstrate for the first time that life-long quiescent stem cells retain the potential to become activated and develop into specific types of brain cells. The six-year long-term neural stem cells are an excellent model for studying developmental neurobiological processes and aging.

Target specific optimization of cationic lipid-based systems for pulmonary gene therapy.

PURPOSE: Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung. METHODS: The effect of varying the surface and colloidal properties of cationic lipid-based gene delivery systems was assessed by intratracheal instillation into rats. An expression plasmid encoding chloramphenicol acetyl transferase (CAT) was used to measure transgene expression. RESULTS: Cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and topology of the plasmid, were found to significantly affect transgene expression. Complexation with lipids was found to have a protective effect on DNA integrity in bronchoalveolar lavage fluid (BALF). DNA complexed with lipid showed enhanced persistence in rat lungs as measured by quantitative polymerase chain reaction. CONCLUSIONS: Fluorescence microscopy analysis indicated that the instilled formulation reaches the lower airways and alveolar region. Data also suggests cationic lipid-mediated gene expression is primarily localized in the lung parenchyma and not infiltrating cells isolated from the BALF.

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).

Consequences for fetus and neonate of maternal red cell allo-immunisation.

AIMS: To study the distribution of clinically important red cell antibodies in pregnancy, and the associated fetal and neonatal morbidity and mortality. METHODS: The case notes of women with clinically important red cell antibodies identified in their serum during pregnancy were reviewed. RESULTS: During a 12 month period 22,264 women were referred for antenatal screening. Clinically important red cell antibodies were detected in 244 (1%). Of these, 100 were anti-D and 144 were non-RhD antibodies. There were three intrauterine deaths, three fetuses required intrauterine transfusion, 10 neonates were transfused, 27 others had phototherapy, and 27 with a positive direct antiglobulin test received no treatment. Early fetal losses occurred in the presence of both high and low levels of anti-D. CONCLUSIONS: Anti-D remains the most common clinically important antibody in pregnancy, and accounts for the greatest fetal and neonatal morbidity and mortality. Of the other antibodies detected, anti-c was associated with most neonatal morbidity. The production of many of the non-D antibodies detected could be avoided by the use of selected red cells when transfusing pre-menopausal women.

Synthetic peptide-based DNA complexes for nonviral gene delivery.

A major advantage of synthetic peptide-based DNA delivery systems is its flexibility. By design, the composition of the final complex can be easily modified in response to experimental results in vitro and in vivo to take advantage of specific peptide sequences to overcome extra- and intracellular barriers to gene delivery. The extreme heterogeneity which greatly complicates both the kinetics of DNA-poly(L-lysine) interaction and the thermodynamic stability of the final DNA complexes is avoided. Other unique features include the absence of biohazards related to the viral genome as well as the production of the viral vector and the absence of limitations on the size of the therapeutic genes that can be inserted in the recombinant viral vector. In principle, if the gene can be cloned into an expression plasmid, it can be delivered as a synthetic DNA complex. Since these synthetic delivery systems are composed of small peptides which may be poorly antigenic, they hold the promise of repeated gene administration, a highly desirable feature which will be important for gene targeting in vivo to endothelial cells, monocytes, hepatocytes and tumor cells.

Measurement of feto-maternal haemorrhage: a comparative study of three Kleihauer techniques and tow flow cytometry methods.

In the UK a Kleihauer test is routinely performed on all RhD-negative women after the birth of an RhD-positive child to ensure that an adequate dose of anti-D immunoglobulin is given. The results of Kleihauer testing are interpreted to assess the volume of any feto-maternal haemorrhage and additional anti-D immunoglobulin is administered if necessary. A similar procedure is followed ante-natally when incidents occur known to be associated with alloimmunization. The performance of Kleihauer tests is difficult to standardize and there can be problems in interpreting the volume of feto-maternal haemorrhage. The use of flow cytometry to measure feto-maternal haemorrhage is reported to give more accurate and reliable results. This study compared three different Kleihauer methods and two different flow cytometry techniques all performed using the same maternal sample and within a single laboratory. The results demonstrated variability between the Kleihauer tests used and also in the flow cytometry measurements. A well-performed Kleihauer test would still appear to be useful as a screening technique for detection of feto-maternal haemorrhage. However, accurate quantitation of size of feto-maternal haemorrhage is more reliably determined by flow cytometry.