Journal of Intellectual Disability Research. Volume 37, Part 6.
December 1993. England.
L. C. AGUILAR, A. ISLAS, P. ROSIQUE, B. HERNÁNDEZ, E. PORTILLO, J. M. HERRERA, R. CORTÉS, S. CRUZ, F. ALFARO, R. MARTIN Y J. R. CANTÚ.
CONTENTS
• Abstract
• Introduction
• Materials and Methods
• Results
• Discussion
• References
ABSTRACT
Basic fibroblast growth factor (bFGF) has shown a neurotrophic effect in the neurons of several CNS ares.
IN VIVO, it contributes to restore neurochemical and morphological deficits in different rodent models of brain damage, including rats with brain damage induced by hypoxia/ischemia when FGF was intramuscularly (IM) administered.
Toxicological and inmunological studies performed in rats, mice and volunteers showed no evidence of side-effects.
Bovine FGF was intramuscularly (IM) administered in children with mental retardation caused perinatal hipoxya, aged 1-15 years, at dosages of 0.4 or 0.28 µg/kg, once or twice a month, over 7-12 months.
Group A [n=12; 6 treated (T), 6 controls (Ct)], group B (n=16; 8T, 8Ct) and group C (n=67; 45T, 22Ct) were evaluated with the P.A.R. scale, the WISC-RM and the Gesell scale, respectively.
Development increased significantly in treated children from groups A ( p < 0.02 ) and C ( p < 0.001 ), and IQ rose by more than 10 points ( p < 0.001 ) in group B patients.
INTRODUCTION
Basic (b) fibroblast growth (FGF) enhances the survival and neurite outgrowth of several CNS fetal neurons (Walicke et al. 1986; Morrison et al. 1986; Walicke et al. 1988; Ferrari et al. 1989; Matsuda et al. 1990) and postnatal neurons (Matsuda et al. 1990), and bFGF also increases the mitogenic activity on astrocites (Morrison & de Vellis 1981) and oligodendrocites (Saneto & de Vellis 1985).
IN VIVO, it has been demonstrated that bFGF prevents the death of septal cholinergic neurons after damage (Anderson et al. 1988), and reverses the morphological and chemical deficits of strital dopaminergic neurons of MPTP-treated mice (Otto & Unsicker 1990).
The acidic (a) FGF shows partial recovery in young MPTP-treated mice of the nigrostriatal dopaminergic system (Date et al. 1990).
Previous studies (unpublished) carried out by our group in Wistar rats wits brain damage induced by hypoxia/ischemia have shown that intra muscular bovine FGF prevents the decrease of dopamine in striatum, and choline acetyltransferase activity in striatum and hippocampus, also significantly improving some neurophysiological parameters (frequency analysis and visual evoked potentials). The treatment of mental retardation (MR) and other cerebral dysfunctions is mainly concentrated on the control of convulsive disorders and hyperkinesia, whereas therapies to improve development and to enhance cognitive capabilities are usually by physical methods.
The present authors report the obtention, characterization, toxicological and inmunological evaluation of bovine FGF, and the results of FGF therapy in children with MR due to brain damage caused by perinatal hypoxia, who were evaluated by different psychometric tests
MATERIALS AND METHODS
• FGF Obtention
• In Vitro Studies
• Enzyme-linked immunosorbant assay
• Toxicological evaluations
• Genotoxicity
• Immunological evaluations
• Tolerance and immunological evaluations in volunteers
• Clinical evaluations
• Therapeutic programme
• Statical analysis
FGF obtention
Fetal bovine brains (5-6 months of gestation) obtained in sterile conditions were homogenized and centrifuged at 5000 g at -5 degree Centigrade for 30 min.
The supernatant was collected and filtered in a pellicon cassette system (om-41 Millipore *) through membranes of 0.2 µm, and 100 and 30 kDa, and then it was concentrated with a 10-kDa membrane.
It was the injected into an HPLC (Delta prep 3000 Waters *) with a prepak C18 column and eluted with 0.1 % trifluoroacetic acid (TFA) flowing at 100 ml/min.
Detection was performed in a spectrophotometer (Waters * Lambda-max mod.481) at 215 nm; the data were integrated in a Data Module (Waters * 745).
All fractions were analyzed for protein quantification by the method of Lowry et al. (1951), and l0% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to assess the MW ranges.
Analytical studies were performed by HPLC with a Bondapak c-18 column (3-9 X 300 mm) using FGF (Sigma *) as reference, and a solution consisting of 80% of TFA (0.1%) and 20% acetonitrile as eluents, flowing at 1 ml/min to assess retention time (RT) and area (BC).
In vitro studies
The biological activity of the FGF obtained after purification was analyzed in neuronal cultures according to Morrison et al. (1986).
FGF was assayed at dosages of 1.0, 0.1, 0.01, 0.005, 0.001 and 0.0005 µg/ml per triplicate at the conversion to chemically defined medium (CDM) and at each medium change.
The optimal dosage was established at 0.005 µg/ml. Cultures with CDM were only used as controls.
Inhibition assays were performed according to Matsuzaki (1989) and Lindner & Reidy (1991) by pre-incubating (10 min, 37 degree Centigrade) 3 µg/ml of monoclonal antibodies antibovine aFGF, and/or 3 µg/ml of antibovine bFGF (Promega Biotec *), with 0.3 µg/ml of the FGF obtained or 0.3 µg/ml of commercial nerve cells as described above (2.5 X 10 E5 per 35-mm well).
Enzyme-linked immunosorbant assay
In order to characterize the FGF, an ELISA was performed by placing the obtained FGF in separate plastic wells.
They were then incubated for 1.5 h with a 1:2000 dilution of rabbit anti-bovine aFGF or anti-bovine bFGF (Sigma *), then they were washed and incubated with a second peroxidase conjugated goat anti rabbit IgG (Sigma *) for 1 h and washed again three times; substrate (diaminobenzidine, Sigma *) was added in 10 ml of PBS 20 µl of oxygenated water and 70 µl of a 1% calcium chloride solution until the development of color, and the reaction was stopped with sulfuric acid.
Toxicological evaluations
Fraction IV (see Results), identified as FGF, was evaluated to ascertain the lethal dosage 50 (LD 50).
Intra muscular (IM) and intra peritoneal administration of FGF to different groups of male BALB/c mice (n=10) at dosages of 60, 600, 1800 and 3000 µg/kg, and 500 mg/kg was followed by measurements of glucose daily for 2 weeks and body weigth 2 times per week.
In order to test for possible chronic toxic effects of FGF, such as neoplasia and hiperplasia, a group of mice (n=10) was treated with 500 mg/kg (IM) every 3 days from birth until the age of 13 months; two more groups were used as controls, one received saline solution as a placebo and the other was not manipulated.
A new group of Wistar rats (n=10) was treated daily with 600 µg/kg (IM) over 2 months. (br) Hemogram, glutamic oxalacetic transaminase, glutamic piruvic trasaminase, uric acid, urea, creatinin, urine general examination (pH, albumin, hemoglobin, lipids and sediment), body weight and survival, were evaluated every week.
At the end of experiment, both groups were sacrificed; necropsy and histopathological analysis were performed.
A further experiment was carried out in two groups (n=6 each) of pregnant rats, one group receiving FGF daily 300 µg/kg (IM) during the whole pregnancy, whereas the other receiving only saline solution; the products were analyzed for teratogenic changes.
Genotoxicity
The Ames test was used to analyze the mutagenicity and carcinogenesis potential of FGF, according to Maron & Ames (1983).
Four strains of Salmenella thyphimusrium (TA 97A, TA 98, TA 100 and TA 102) were exposed to different concentrations of FGF (4.8, 3.5, 1.2 and 0.6 µg/ml) with and without metabolic activation, negative control (which received the water used as solvent), and positive control (mutagenic product) were used as references.
The results were expressed according to the number of colonies per plaque in duplicate assays; a mutagenic event was considered when the number of colonies per plaque were double or more than that of the negative control and showed the presence of a dose-response behavior.
Immunological evaluation
Five groups (n=5) of Wistar rats were formed. The first group was used as the control, receiving only saline solution.
A second group (positive controls) received 1.6 mg/kg (IM) of the supernatant from total extracts of fetal bovine brain (initial FGF isolation stage).
The third group received 16 mg/kg of the supermatant.
The fourth group received 30 µp/kg (IM) of FGF (final stage), a dosage higher than estimated useful to improve neurochemical and neurophysiological parameters in previous studies performed in rats with brain damage.
The fifth group received 300 µg/kg (IM).
All applications were given twice every week.
After 4, 8, 12 and 16 weeks, serum was obtained to quantify antibodies against FGF using an ELISA test as described above, but now using the rat sera in order to search for the possible presence of antibodies and goat anti-rat immunoglobulin as a second peroxidase linked antibody.
Tolerance and immunological evaluation in volunteers
In order to test the tolerance and immunological safety of bovine FGF, eigth healthy adult volunteers received 0.4, 0.28, 0.14 and 0.07 µg/kg every 2 weeks for 6 months, and their sera were analyzed every month by the ELISA described above with a minor modification (the second peroxidase linked antibody was against human gamma globulin).
Paraclinical studies (blood cell count, blood chemistry and urine analysis) were performed in each indivudual 3 and 6 months after the first administration.
Clinical evaluation
Nineth-five children with MR, who showed a generalized lag in development and antecedents of perinatal hypoxia and had devepmental and intellectual quotients (DQ an IQ, respectively) of less than 60, in whom the hypoxia was considered the most feasible aetiology (other causes were excluded by clinical and laboratory tests), and without any other chronic or acute disease, epilepsy or paroxismal activity, were included in this study.
Various abnormalities in neurophysiological parameters (magnitude and symmetry of absolute and relative power, linear correlation and magnitude of energy of topographic visual evoked potentials) were observed in all patients.
All the children were living with their families.
The parents of all patients were thoroughly informed about the details, results of the studies describes above and the potential risks of the investigation before granting their approval to participate in a double-blind research study.
It should be made clear that all preclinical studies required by the general law of health in matters pertaining to the research of new drugs (Reglamento de la Ley General de Salud en Materia de Investigacion para la Salud 1990) were fulfilled before the clinical studies.
Group A consisted of children (4 males, 3 females), aged 1-4 years, with MR (DQ=30) attending an early intervention service of a special education school in Chihuahua, Mexico, were evaluated at the beginning and end of a 10-month therapeutic period using the official developmental scale of The Mexican Ministry of Education (based on the psychogenetic theory of Jean Piaget), and routine paraclinics (blood cell count, blood chemistry and urine analysis).
A control group was matched in number, age, sex, and level of MR. Both groups belonged to the same class and werte under the same type of educational stimulation.
Group B was formed of eight children (3 males, 5 females) with MR (IQ=50), aged 6 to 11 years, who were enrolled in a special education school in Guadalajara, Mexico, for children repeating at least twice the same grade at elementary school.
A control group (number, age, sex and level of MR matched) of patients from the same class and receiving the same type of stimulation was formed.
Patients froms both groups were evaluated at the beginning and end of a 7-month therapeutic period with the following tests: WISC-R (Mexican version), Bender visual-motor-gestalt test, and routine paraclinics.
Group C consisted of 45 treatd and 22 control patients all evaluated with Gesell scale (DQ=30).
Three subgroups were formed according to age: Cl from 0 to 4 years (n=22, 12 males, 10 females), with a control group (n=9,5 males, 4 females), age and level of MR matched; C2 from 5 to 8 years (n=18, 10 males, 8 females), C3 with ages between 9 and 15 years (n=5,4 males, 1 female) with a control group matched in number, age, sex and level of MR.
Therapeutic programme
The dosage per application of bovine FGF (0.4 µ:g/kg of body weight) was used in groups A and B, with one single application per month, and in group C(0.28 µg/kg of body weigth) every 2 weeks.
The duration of treatment in each group was: 10 months for A, 7 months for B and 12 months for C.
Immunological studies identical to those in volunteers were performed in each patient every month during the first 3 months if therapy, and then every 3 months until the end of the study.
Statical analysis
The cell cultures results were analyzed by a t-test.
In the children of groups A, B and C, the differences of development or IQ v between the first and second or third evaluation (group C) were totalized by group and compared to their respective controls by a t-test’ the Mann-Whistney U.Wilcoxon rank test was also used in groups A and B.
RESULTS
• FGF insolation
• In vitro studies
• Toxicological studies
FGF insolation
The fraction with RT=145.92 min in preparative chromatogram [named as fraction IV (F-IV); (Fig. 1a)] show in the HPLC analytic study two peaks with RT=6.89 and 7.66 min (Fig. 1b), which was very similar to the RT (6.83 and 7.6) of reference FGF (Sigma *) (Fig. 1c), mainly as bFGF and a small part as aFGF.
Different eluent concentration (acetonitril and TFA) did not show other peaks.
SDS-PAGE revealed a single band in F-IV with a MW of 16 kDa in different trials.
In vitro studies
FGF (F-IV) induced a significant ( p < 0.001 ) increase in cell survival (sixth day) and the number of process-bearing cells (third and sixth days), the inhibition assays adding monoclonal antibodies against bFGF to F-IV (FGF) or to reference FGF, showed that the survival (data not shown) and process-bearing cells were significantly decreased when compared to FGF (F–IV) or commercial (reference) FGF alone (Fig. 2).
When the monoclonal anti-aFGF was added to FGF (F-IV) or reference FGF cultures, the survival and process-bearing cells were not inhibited.
The ELISA test revealed the presence of bFGF and aFGF when anti bFGF or anti aFGF were used as first antibody in both cases, FGF (F-IV) and reference FGF, in dose-response behavior (fig 3).
Toxicological studies
The results did not allow the establishment of a LD 50 since no death occurred a 500 mg/kg, which was the maximum dose used in this experiment.
Survival, body weight and glucose parameters showed no differences between treated and control groups.
The chronic (long-term) evaluation yielded no significant changes in either the BALB/c mice or the Wistar rats experiments, as compared to controls; the necropsy and histopatological analysis did not show any increment of volume or weight, hyperplasias, or neoplastic changes in organs and tissues.
No teratogenic changes were observed in the products of treated pregnant rats.
The results of the Ames test in assays with and without metabolic activation revealed no mutagenicity of FGF.
The ELISA tests performed in rats (Table 1) and human volunteers did no detect antibodies against bovine FGF in any case.
The paraclinical tests did not show any significant findings. No side-effects besides occasional mild headaches were observed in volunteers.
It is wort mentioning that all volunteers reported that they had experienced more vital energy (e.g. awareness, libido and physical fitness), and some of them irritability.
As shown in (Fig. 4), there was a significant developmental improvement in children of group A with respect to controls.
In group B, an increase of about 10 points in (Fig. 5). The Bender test revealed and increase in development of 6 months in the treated group, whereas the control group showed no increase at all.
However, no significant statistical differences were found most likely due to dispersion.
In groups C1 and C2, a significant increase ( p < 0.001 ) in the development of the treated patients was found (Fig.6a,b).
Because of the small number of cases in group C3, each one was evaluated separately and the results described individually (Fig.6C); the increase in the rate of development of the treated groups modified the development quotient (DQ) as follows; 9.44% of increase in C1 ( p < 0.001 ), 5.67% in C2 ( p < 0.01 ) at 12 months and l.2% in C3 (n.s.) at 6 months as compared to controls.
The ELISA tests performed in patients did not detect antibodies against bovine FGF in any case.
The paraclinical tests did not show any abnormal finding in patients or controls.
About 10% of the treated patients showed a slight hyperactivity and irritation during the first two or three applications. No other side-effect was observed.
DISCUSSION
The fraction IV was considered to be bFGF and aFGF, based on the following data.
The RTs of F-IV and reference FGF were practically the same, MW (16 kDa) of F-IV observed in the SDS-PAGE is also similar to the MW of bFGF and aFGF reported by Baird elt al. (1986) and to the MW of reference FGF.
The ELISA for FIV showed immunoreactiovity in a dose-response behavior when antibodies versus aFGF and bFGF were used, thus confirming their presence.
The in vitro results of neurotrophic activity of FGF (F-IV) on cultured cortical cerebral cells were similar to those reported by Morrison et al. (1986) using bFGF.
The percentage of glial cells was not determined by specific markers, but according to the data reported by Morrison et al. (1986), it could be estimated as between 3 and 7%.
This neurotrophic effect was blocked by monocional antibodies against bFGF, thus corresponding that F-IV was indeed mostly bFGF.
The in vivo stimulating effect of (IM) administration of F-IV to rats with brain damage induced by hipoxia/ischemia on cholinergic and dopaminergic neurons observed by the present authors (unpublished data) was similar to the one observed after local applications of bFGF (Anderson et al. 1988; Otto & Unsicker, 1990), and similar to local applications of aFGF to dopaminergic neurons (Date et al. 1990).
Furthermore, the analytical chromatogram of F-IV, according to the information supplied by Sigma * on the FGF used as reference, suggests that FGF (F-IV) is formed mainly by FGF (71.08%) and a minor quantity of acidic FGF (28.83%) with a purity approximately 99%.
The toxicological evaluation failed to detect abnormalities in the FGF-treated groups.
The LD 50 could not be obtained at dosages even 20000 times higher than those considered useful no treat rats with brain damage successfully.
These results are similar to those reported by Mazue et al. (1991), who tested recombinant human bFGF in different animal models, and found an enormous difference between the useful and the lethal dosages.
The chronic administration of very high dosages during long-term periods fails to detect abnormalities including neoplasias, hyperplasias and abnormal proliferation in vascular and skin tissues, probably due to a low expression of high-affinity receptors in healthy conditions.
The application of bovine FGF to volunteers over 6 months showed a good tolerance and no immunological response.
As far as development is concerned, the recovery of patients, quantified by three different tests, suggests that it is related to the effect of FGF on development, regeneration and synaptic plasticity of central neurons (Seifrt et al.1990).
Many cellular mechanisms similar to those that operate during development continue to be available to the adult and are actualized when necessary (Cotman & Nieto-Sampedro, 1985).
It seems that the FGF potentializes this capacity.
After the first 6 months of FGF therapy, the rate of development was found to be higher in groups C1 and C2 than after the second 6-month period.
This is probably due to the expression of inhibitory factors on innervation (Goldowitz & Cotman, 1980), or to the down regulation of FGF receptors which are normally expressed in adult CNS (Wanaka et al. 1990).
The expression of bFGF in normal CNS suggests a quotidian role in its function (Cordon-Cardo et al. 1990); it increase after strike (Finklestein et al. 1990) and after the injury of the nervous system (Eckenstein et al. 1991), suggesting that FGF is necessary for the restoration of neuronal function after damage.
The most widely held view al the present time is a supposed total impermeability of the blood-brain barrier (BBB) to peptides in general (Meisenberg & Simmons 1983; Pardridge 1984).
The present authors’ group have recently developed experiments in rats that received intravenous I-131 FGF which show the presence of radioactivity after 25 min in the brain parenchyma, with a previous detection in cerebral capillaries at 5 min, suggesting an active transport by endothelial cells of cerebral capillaries since they have receptors to bFGF (Schweigerer et al. 1987).
This mechanism is probably similar either to the receptor-mediated transcytosis of transferrin (Fishman et al. 1987).
The effect of FGF (F-IV) after (I) administration to rats with CNS damaged by hypoxia-ischemia and the present results in children with MR due to perinatal hipoxia confirm that FGF could access the CNS.
The present way of administration, (I), can contribute to reduce risks of local administration in the brain in future treatments of brain damage and neurodegenerative disorders.
The lack of antibody formation against bovine FGF in rats, volunteers or patients is probably associated to the minimal amount of FGF used, its purity (99%), the low administration frequency and high homology (98.7%) between human and bovine bFGF (Abraham et al. 1986), which is higher than the one between bovine and human insulin.
Given FGF effectiveness on partially recovering the lag in development in children with MR,its lack of toxicity in several animal experimental models, the absent immunogenicity (at least at the dosages and the therapeutic periods here analyzed), the minor side-effects, and the convenient way of administration, it seems reasonable to consider that this therapeutic approach for MR due to brain damage is at least promising as an alternative to help these patients.
Certainly, FGF therapy deserves much more attention and research.
The administration of FGF could be hazardous in proliferative disorders, like gliomas and arterial diseases presenting abnormal endothelial cell proliferation.
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31. Received 23 july 1992.
