Cannabidiol (Epidyolex®) for severe behavioral manifestations in patients with tuberous sclerosis complex, mucopolysaccharidosis type III and fragile X syndrome: protocol for a series of randomized, placebo-controlled N-of-1 trials.

BACKGROUND: Many rare genetic neurodevelopmental disorders (RGNDs) are characterized by intellectual disability (ID), severe cognitive and behavioral impairments, potentially diagnosed as a comorbid autism spectrum disorder or attention-deficit hyperactivity disorder. Quality of life is often impaired due to irritability, aggression and self-injurious behavior, generally refractory to standard therapies. There are indications from previous (case) studies and patient reporting that cannabidiol (CBD) may be an effective treatment for severe behavioral manifestations in RGNDs. However, clear evidence is lacking and interventional research is challenging due to the rarity as well as the heterogeneity within and between disease groups and interindividual differences in treatment response. Our objective is to examine the effectiveness of CBD on severe behavioral manifestations in three RGNDs, including Tuberous Sclerosis Complex (TSC), mucopolysaccharidosis type III (MPS III), and Fragile X syndrome (FXS), using an innovative trial design. METHODS: We aim to conduct placebo-controlled, double-blind, block-randomized, multiple crossover N-of-1 studies with oral CBD (twice daily) in 30 patients (aged ≥ 6 years) with confirmed TSC, MPS III or FXS and severe behavioral manifestations. The treatment is oral CBD up to a maximum of 25 mg/kg/day, twice daily. The primary outcome measure is the subscale irritability of the Aberrant Behavior Checklist. Secondary outcome measures include (personalized) patient-reported outcome measures with regard to behavioral and psychiatric outcomes, disease-specific outcome measures, parental stress, seizure frequency, and adverse effects of CBD. Questionnaires will be completed and study medication will be taken at the participants' natural setting. Individual treatment effects will be determined based on summary statistics. A mixed model analysis will be applied for analyzing the effectiveness of the intervention per disorder and across disorders combining data from the individual N-of-1 trials. DISCUSSION: These N-of-1 trials address an unmet medical need and will provide information on the effectiveness of CBD for severe behavioral manifestations in RGNDs, potentially generating generalizable knowledge at an individual-, disorder- and RGND population level. TRIAL REGISTRATION: EudraCT: 2021-003250-23, registered 25 August 2022, https://www.clinicaltrialsregister.eu/ctr-search/trial/2021-003250-23/NL .

Morphological and functional changes due to drug-induced lysosomal storage of sulphated glycosaminoglycans in the rat retina.

UNLABELLED: A series of dicationic amphiphilic drugs, most of them immunomodulatory agents, are known to induce generalised lysosomal storage of sulphated glycosaminoglycans (GAGs) in rats and in cultured cells of several species including man. The present study deals with the cytological effects of two experimental immunomodulatory acridine derivatives upon the retina of rats. The animals were treated orally with compound CL-90.100 (3,6-bis[2-(diethylamino)ethoxy]acridine) or an analogue for periods up to 22 weeks at a dose range of 60-90 mg/kg body weight and the retinae examined by light and electron microcopy. ERG measurements were done initially and after 16 weeks of treatment. All types of retinal cells developed abnormal cytoplasmic vacuoles which represented the ultrastructural counterpart of lysosomal GAG storage as demonstrated by histochemical and cytochemical staining experiments. The retinal pigment epithelium and the Müller cells were most prominently affected, photoreceptor cells to a lesser degree, and retinal neurons to varying degrees. The topographical distribution of the drug as detected by fluorescence microscopy closely resembled the distribution of the GAG accumulation in the retinal layers. After treatment for 16 weeks, the a-and b-wave amplitudes in the ERG were significantly reduced compared with the controls. CONCLUSION: the glycosaminoglycan storage in pigment epithelium is reminiscent of that seen in some inherited mucopolysaccharidoses of humans. When a given cell type shows lysosomal accumulation of glycosaminoglycans as a consequence of impaired degradation, it can be assumed to be engaged in the turnover of glycosaminoglycans under normal conditions. Thus the present results suggest that not only the retinal pigment epithelium but also Müller cells, photoreceptor cells, and, to variable degree, retinal neurons are normally involved in the catabolism of sulphated glycosaminoglycans. We believe that the lysosomal storage of glycosaminoglycans caused secondary cellular disturbance responsible for the functional changes shown by electroretinography.

The antimalarials quinacrine and chloroquine induce weak lysosomal storage of sulphated glycosaminoglycans in cell culture and in vivo.

The antimalarial agents quinacrine and chloroquine are well known as potent inducers of lysosomal storage of polar lipids (lipidosis) in cell culture and in vivo. In previous experiments on cultured fibroblasts, chloroquine was shown to additionally cause weak lysosomal storage of sulphated glycosaminoglycans (GAGs) thus inducing mucopolysaccharidosis (MPS). In the present study, quinacrine was investigated for this ability, because we wished to know whether or not the acridine ring system in quinacrine would enhance the MPS-inducing potency as compared to chloroquine carrying an isoquinoline ring system. Tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one) known as a potent inducer of MPS served as reference compound. The compounds were compared at a concentration (3 microM) which did not enhance the secretion of the lysosomal enzyme beta-hexosaminidase (E.C. 3.2.1.52), since this would be an indication of unspecific drug effects upon the endosomal/lysosomal compartments of the cell. Additionally the liver of quinacrine- and chloroquine-treated rats was examined with the question whether the lysosomal GAG storage induced by either drug in cell culture had an equivalent in intact organisms. Both, in cell culture and in vivo, quinacrine was found to be a more potent inducer of lysosomal GAG storage than was chloroquine. The results suggest that the acridine ring system favours this drug side effect as compared with the bicyclic isoquinoline ring system. On the other hand, quinacrine was significantly less potent than tilorone and the Symmetrically substituted acridine derivative 3,6-bis[2-(diethylamino)ethoxy]acridine investigated previously. This suggests that the asymmetric structure of the quinacrine molecule reduces the potency as compared to the symmetrically substituted bisbasic compounds with planary tricyclic ring systems such as tilorone and congeners.

Lysosomal storage of sulfated glycosaminoglycans induced by two bis-aminomethyl anthrachinones.

Several immunomomodulatory drugs, all of them symmetrically substituted dicationic amphiphilic compounds, are known to cause lysosomal storage of sulfated glycosaminoglycans (GAGs) in intact animals and cultured fibroblasts. The storage is due to impaired GAG degradation. The standard compound is tilorone (2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one). In the present study two bis-aminomethyl anthrachinones were examined for their ability to induce lysosomal GAG storage in cultured bovine corneal fibroblasts. For reference, a bis-aminoethoxy-anthrachinone compound (RMI-10.024) was included, which is known to be a potent inducer of lysosomal GAG storage. The present morphological, radiochemical, and biochemical results show that the bis-aminomethyl anthrachinone compounds investigated cause lysosomal storage of GAGs, although with significantly lower potencies than the bis-aminoethoxy anthrachinone. Dermatan sulfate contributed approximately 90% to the drug-induced increment of intracellular GAGs. The present results suggest that the length of the side chains, i.e., the distance between the aromatic ring system and the protonizable nitrogen of the side chains, and the position of the side chains relative to the aromatic ring system are important molecular features influencing the potency of inducing lysosomal GAG storage.

Suramin-induced mucopolysaccharidosis in rat incisor.

Two weeks after a single injection of suramin, the secretory and post-secretory ameloblasts of the rat incisor were filled with large lysosome-like vacuoles. At the light-microscope level, these vacuoles were positively stained with Alcian blue when MgCl2 was used at a critical electrolyte concentration varying between 0.1 and 0.3 M, whereas no staining appeared when MgCl2 varied between 0.7 and 0.9 M. Hyaluronidase digestion markedly reduced but did not totally abolish the staining, indicating that glycosaminoglycans were accumulated inside these vacuoles. Examination of these cells with the electron microscope revealed a polymorphic population of large vesicles, filled to various degrees with cetylpyridinium chloride (CPC)-positive and malachite green aldehyde (MGA)-positive material. The same pattern was observed in secretory odontoblasts but to a lesser extent. In the extracellular matrix, suramin-induced alterations appeared as large defects occurring during enamel formation. In predentin and dentin, the number and/or size of electron-dense aggregates resulting from CPC and MGA fixation, were enhanced in the suramin-injected rats. These aggregates were largely reduced or suppressed respectively by hyaluronidase digestion and chloroform/methanol treatment of the sections. The accumulation of glycosaminoglycans and phospholipids reported here inside ameloblasts and odontoblasts and in predentin and dentin supports the occurrence of suramin-induced mucopolysaccharidosis and lipidosis in this experimental animal model.

[Lysosomal storage diseases, genetic or drug-induced? effect of glycosaminoglycan and sphingolipid disorders on dental tissues]. / Maladies de surcharge lysosomale, génétiques ou induites pharmacologiquement: effets de pathologies de glycosaminoglycanes et de sphingolipides sur les tissus dentaires.

In vivo studies were carried out on dental tissues of rat incisor after a single injection of suramin, a drug which induces mucopolysaccharidosis-like disease. Accumulation of lysosome-like structures was seen in secretory ameloblasts and odontoblasts. In vitro studies on embryonic tooth germ buds showed similar changes when they were cultured in presence of suramin. Anti-phospholipid immunolabelling revealed a developmentally regulated temporo-spatial pattern. Radiolabeling with 3H-suramin indicated cytosolic and nuclear incorporation. The drug acting as polyanion interacted directly with predentine. 35S sulphate incorporation was impaired by the drug. Another lysosomal storage disease, the sphingolipidosis, Krabbe's disease was also investigated in human. Changes were observed in pulp cells and as a consequence in dentin. Enamel also displayed many changes. Pharmacological or genetically acquired diseases constitute models providing insights on the role played by glycosaminoglycans and phospholipids in biomineralization.

Induction of mucopolysaccharidosis in rats by treatment with immunostimulatory acridine derivatives.

In the accompanying paper, four dibasic acridine derivatives were reported to induce lysosomal storage of sulfated glycosaminoglycans (sGAG), i.e., mucopolysaccharidosis, in cultured fibroblast (Handrock et al. Toxicol. Appl. Pharmacol. 114, 1992). The purpose of the present morphological and biochemical investigation was to examine whether two representatives of the acridine derivatives, namely 3,6-bis[2-(diethylamino)ethoxy]-acridine and the piperidino analogue, induce mucopolysaccharidosis in intact organisms. Rats were orally treated with 60-80 mg/kg up to 22 weeks. Morphological examination of liver, spleen, and blood lymphocytes yielded cytochemical evidence of mucopolysaccharidosis. Biochemically, up to a 48-fold increase of the urinary excretion of sGAG was found. In the liver and spleen of chronically treated rats, the sGAG contents were elevated by factors up to 56 and 23, respectively. Heparan sulfate and dermatan sulfate contributed most to the total increase of sGAG; chondroitin sulfate was stored to a minor degree. For one compound, the tissue concentrations were determined. It was found that the drug was accumulated in the tissues. Due to their fluorescent properties, the drugs could be detected by fluorescence microscopy to be present in high concentrations within the sGAG-storing lysosomes. On the basis of these observations and of the biochemical data it appears justified to assume a ratio of at least one drug molecule per disaccharide unit of the sGAG to be present in the lysosomes. It is proposed that this leads to the formation of sGAG-drug complexes in the lysosomes. Such complexes may be indigestible substrates for the lysosomal enzymes, thus leading to mucopolysaccharidosis. For toxicologic practice, the cytochemical examination of lymphocytes is recommended as a simple measure for early detection and monitoring of this adverse drug effect.

Osteopenia in rats with drug-induced mucopolysaccharidosis.

The immunostimulatory drug tilorone (CAS 27591-97-5) induces generalized lysosomal storage of sulfated glycosaminoglycans, i.e., mucopolysaccharidosis (MPS) in rats. Since inherited MPS of man is often associated with skeletal lesions the present study was performed to investigate whether the tilorone-induced MPS is also accompanied by bone alterations. The answer is yes. Rats (initial age 4 weeks) were orally treated with tilorone (60-80 mg/kg; 6-25 weeks). The proximal tibial metaphysis was examined by radiography and by light and electron microscopy. The tibiae of drug-treated rats remained smaller than those of pair-fed control rats. The primary bone trabecules in drug-treated rats became increasingly shorter, and the cortical bone became thinner. The general organization of the cartilaginous growth plate and the zones of vascular invasion and primary trabecule formation appeared similar as in control rats. Increased numbers of active osteoclasts were found at the free ends of the primary trabecules. Ultrastructurally, MPS-like alterations were observed in all types of bone cells. The osteopenia was partly reversible during recovery periods of more than 6 months after discontinuance of drug treatment. At present, the causal relationship between drug-induced MPS and osteopenia, as well as the mechanisms responsible for osteopenia are unknown. It is suggested that the principal factors are stimulation of osteoclasts and retardation, though not profound distortion, of the events involved in enchondral ossification.

The tilorone-induced mucopolysaccharidosis in rats. Biochemical investigations.

The tissues of rats chronically treated with tilorone exhibited a significant accumulation of acid glycosaminoglycans (GAGs): In the liver, the GAG concentration was found to be elevated by a factor of 38, in the spleen by a factor 15 and in the kidneys by a factor of 5. Furthermore, the renal excretion of GAGs was increased 32-fold as compared to control animals. Dermatan sulphate was predominant among the GAGs stored in the three organs; chondroitin sulphate and heparan sulphate were found in smaller amounts. GAG storage was accompanied by accumulation of the drug within the tissues: the molar ratio of tilorone per disaccharide unit of GAG was calculated to be one to two in each tissue. According to previous reports, tilorone-induced mucopolysacchariodosis is due to impaired lysosomal degradation of GAGs. The present results give support to the hypothesis that an interaction between the polyanionic GAGs and the dicationic drug may lead to GAG-drug complexes which cannot be digested by lysosomal enzymes.

Acridine Orange, a precipitant for sulfated glycosaminoglycans, causes mucopolysaccharidosis in cultured fibroblasts.

The purpose of the present investigation was to examine whether or not a di-cationic amphiphilic compound that is known (1) to be accumulated in lysosomes and (2) to form insoluble complexes with sulfated glycosaminoglycans (sGAG) in vitro, is able to interfere with the lysosomal degradation of sGAG, thus causing mucopolysaccharidosis (MPS) in cultured cells. Acridine Orange (AO) was chosen for this study since it is known to meet the above requirements. Cultured fibroblasts from rat cornea were exposed to AO (0.7 microM to 30 microM) for 72 h; tilorone served as reference compound. AO (1.75 microM to 10 microM) caused MPS in a concentration-dependent manner, higher concentrations were cytotoxic. MPS was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The sGAG-complexing properties of AO were demonstrated by using it as a fixative for the intralysosomal sGAG accumulated in tilorone-treated cells. The present findings give support to the working hypothesis that the MPS induced by di-cationic amphiphilic drugs is due to the formation of insoluble sGAG-drug complexes, with the result that the sGAG become resistant to lysosomal degradation.

Human accumulation potential of xenobiotics: potential of catamphiphilic drugs to promote their accumulation via inducing lipidosis or mucopolysaccharidosis.

1. Drug accumulation without a concomitant elevation of blood level may occur if the capacity of the tissue to bind drug increases during chronic treatment. 2. This special type of accumulation is found with cationic-amphiphilic drugs, which induce the formation of lysosomal inclusion bodies containing undergraded lipids or mucopolysaccharides (drug-induced lipidosis or mucopolysaccharidosis, respectively); the stored material provides the additional binding sites for the drug. 3. Factors determining the potential for inducing lipidosis or mucopolysaccharidosis are: (a) affinity of the drugs to phospholipid layers (governed by hydrophobicity) or mucopolysaccharides (drug-induced lipidosis or mucopolysaccharidosis, respectively); the free intra-lysosomal concentration, which is elevated compared with the blood level due to lysosomal trapping (especially with dicationic drugs); (c) the therapeutically required drug concentration in the blood: the therapeutic concentrations are high with drugs that do not act via binding to specific high-affinity receptors.

Mucopolysaccharidosis (MPS) in ocular tissues as induced by amphiphilic di-cationic drugs.

The immunostimulatory drug tilorone (2,7-bis(2- (diethylamino)ethoxy)-9H-fluorene-9-one) has previously been described to induce keratopathy in patients and in rats; in the latter, lysosomal storage of sulfated glycosaminoglycans (mucopolysaccharidosis, MPS) was found to be the reason for corneal clouding. In the present histochemical and ultrastructural study, several ocular tissues of rats were examined after chronic treatment with tilorone and two other immunostimulatory agents (3,6-bis(2-(dipiperidyl)ethoxy)-acridine and the diethylamino analogue). The acridine derivatives hardly affected the cornea and sclera. Tilorone as well as the acridine derivatives induced significant MPS in ciliary body, iris, and choroid. While the functional consequences of drug-induced MPS in ocular tissues are not known yet, except for the cornea, this adverse drug action should be taken into account when new compounds are designed that share certain physicochemical features with tilorone. One molecular feature essential for the MPS-inducing potency of a drug appears to be the dicationic amphiphilic character.

Cultured corneal fibroblasts as a model system for the demonstration of drug-induced mucopolysaccharidosis.

The purpose of the present investigation was to establish a cell culture system suitable for demonstrating the drug-induced lysosomal storage of sulfated glycosaminoglycans (GAGs). This is a drug side-effect which was previously studied in animals treated with the di-cationic amphiphilic compound tilorone and congeners, and which is likely to occur in humans, too. Cultured corneal fibroblasts of rats were exposed to tilorone for 72 h. They developed histochemical and cytochemical alterations indicative of mucopolysaccharidosis and resembling those occurring in vivo. The threshold drug concentration was found to be below 0.7 microM. The reversibility of the lysosomal GAG storage was low. An increase in the drug concentration to 10 microM produced additional unspecific lysosomal alterations, while the mucopolysaccharidosis-like lesions became less prominent. Concentrations of 40 microM and 80 microM caused unspecific cytoplasmic vacuolation and cell death, respectively. The present model system appears suitable for screening investigations of newly developed drugs with respect to their mucopolysaccharidosis-inducing potential and for investigating the structure-activity relationships underlying this adverse drug effect. Care should be taken not to use too high drug concentrations which cause unspecific lysosomal lesions.

Mucopolysaccharidosis and lipidosis in rats treated with tilorone analogues.

The experimental immunomodulatory agent tilorone was previously reported to induce generalized mucopolysaccharidosis (lysosomal storage of sulfated glycosaminoglycans) and lipidosis in rats. While lipidosis is a side effect common to many cationic amphiphilic compounds, none of them except tilorone has been known to cause mucopolysaccharidosis in intact animals. The purpose of the present histochemical and ultrastructural study was to examine whether or not mucopolysaccharidosis can be induced by tilorone analogues. Three analogues were selected and administered to rats in short-term and subchronic experiments, and liver, spleen, kidney, and cornea were examined. The analogues caused generalized cellular lesions which had the same histochemical and cytological characteristics and the same distribution as the lesions produced by tilorone. The results show that the ability to induce mucopolysaccharidosis is not a unique property of tilorone; this drug side effect should be taken into account when developing new drugs with molecular structures resembling that of tilorone.

Experimental mucopolysaccharidosis: preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue.

The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl2 simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.

Mucopolysaccharidosis-like cellular alterations in chondrocytes of rats treated with tilorone.

The dicationic amphiphilic compound tilorone was previously shown to interfere with the lysosomal degradation of sulfated glycosaminoglycans (GAGs) of cultured cells and to cause mucopolysaccharidosis-like alterations in several organs of rats. Since chondrocytes belong to those cells that are severely affected in cases of inherited mucopolysaccharidoses, we wished to know whether this also holds true of the experimentally induced mucopolysaccharidosis. Young rats were treated with tilorone (50-80 mg/kg of body weight) for 2-17 weeks; chondrocytes in tracheal and costal cartilage and in the epiphyseal growth plates of the tibia and ribs were examined by electron microscopy and cytochemistry (acid trimetaphosphatase and staining with Cuprolinic Blue). The tracheal and costal chondrocytes showed numerous abnormal clear vacuoles. Some of them could be identified cytochemically as lysosomes filled with polyanionic storage material, probably sulfated GAGs. In the epiphyseal plate, only the resting chondrocytes were markedly affected, whereas those of the remaining zones were hardly altered; this may be due to the short life span of these chondrocytes in rat growth plates. The present results show that chondrocytes participate in the experimental mucopolysaccharidosis and suggest that, under normal conditions, the lysosomes of chondrocytes have to cope with a considerable load of GAGs.

On the alcianophilia of the drug suramin used as a tool for inducing experimental mucopolysaccharidosis.

The trypanocidal drug suramin was previously reported to induce mucopolysaccharidosis in rats; apart from the biochemical demonstration of increased tissue concentrations of sulfated glycosaminoglycans (GAGs), a strongly positive staining reaction with the cationic dye Alcian Blue was taken as indicating GAG-storage (Constantopoulos et al. 1983). The purpose of the present report is to point out a methodical pitfall. In model experiments it was found that suramin itself, being a polysulfated compound, gives a strongly positive reaction with Alcian Blue at pH 1. It is known that suramin is accumulated in the lysosomes and that high drug concentrations are retained in the tissues for weeks. Therefore a positive staining reaction with Alcian Blue observed in a given cell cannot be conclusively attributed to the storage of sulfated GAGs as has been done in the past. The present report may be a warning that, in the case of the suramin-induced animal model of mucopolysaccharidosis, the usual histochemical strategy, i.e. staining with cationic dyes, is not suitable for analysing the cellular distribution pattern of GAG-storage, since the inducing drug by itself reacts with the indicator dye.

The suramin-treated rat as a model of mucopolysaccharidosis: reversibility of biochemical and morphological changes in the liver.

Rats treated with the trypanocidal drug suramin, a potent inhibitor of several lysosomal enzymes, develop a storage disorder which mimics the features of mucopolysaccharidosis (Constantopoulos et al. 1983). In this paper we have examined the reversibility of the biochemical and pathological changes induced in the liver of the suramin-treated rat. Rats were injected with a single intravenous dose of suramin (250 mg/kg) and allowed to survive for periods of up to 6 months. The liver was examined for suramin content, pathological changes, biochemical storage of glycosaminoglycans (GAGs) and for the blockade of the relevant hydrolytic enzymes. GAG storage in the liver peaked at approximately 14 days after administration of suramin when there was a five-fold increase in the GAG content. Thereafter GAGs decreased in parallel with the fall of suramin concentrations so that within 6 months the content had returned to control levels. The activity of most of the enzymes tested had also returned to control levels within 6 months. The pathological changes which are evident in the liver 1-2 weeks after administration of the drug had diminished considerably within 6 months. These results indicate that significant reversibility of both the biochemical and pathological changes induced by suramin occurs and they support the suitability of the suramin treated rat as a model to assess the value of therapeutic treatments of mucopolysaccharidosis.

Mucopolysaccharidosis-like alterations in cardiac valves of rats treated with tilorone.

The purpose of this study was to examine whether the aortic and mitral valves of rats are involved in the mucopolysaccharidosis-like disorder induced by tilorone. Rats were treated with large doses of the drug for periods of 1-21 weeks. After chronic drug treatment the leaflets of both heart valves were thickened and opaque. In all treated animals the spongiosa layer of the stroma was crowded with vacuolated cells; the fibrosa layer was altered only after prolonged treatment. Ultrastructurally, the vacuolated cells of the spongiosa could be identified as histiocytes and fibroblasts, the former being the most susceptible cell type. The fibroblasts of the fibrosa represented the least sensitive cell type. The histochemical results showed that the clear cytoplasmic vacuoles in the spongiosa cells were due to lysosomal storage of polyanionic material with staining characteristics similar to cartilage matrix. After discontinuation of drug treatment the alterations persisted for several weeks. The present study shows that heart valves are involved in the mucopolysaccharidosis-like disorder induced by tilorone. The molecular pathomechanism of the disorder and the exact identification of the storage material must await further analysis.