Magnitude of Extended-Spectrum Beta-Lactamase-Producing Gram-Negative and Beta-Lactamase-Producing Gram-Positive Pathogens Isolated from Patients in Dar es Salaam, Tanzania: A Cross-Sectional Study.

Background The worldwide emergence of antibiotic-resistant bacteria threatens to overshadow the dramatic advances in medical sciences since the discovery of antibiotics. Antibiotic resistance has rendered some antibiotics obsolete, creating a reliance on synthetic drugs. In some instances, bacteria can be resistant to all antibiotics. The problem of antibiotic resistance is eminent in resource-limited countries like Tanzania, where systematic surveillance and routine susceptibility tests are rarely conducted. Therefore, this study aimed to investigate the magnitude of beta-lactamase-producing Gram-positive pathogens and Enterobacteriaceae with extended-spectrum beta-lactamase (ESBL) in Dar es Salaam, Tanzania. Methodology This multi-site cross-sectional study involved three private hospitals in Dar es Salaam, Tanzania. The study was conducted between July and September 2008. Bacterial isolates were collected, identified, and subjected to antibiotic-sensitivity testing against cephalosporins, including ceftriaxone, cefuroxime and cefotaxime, and clavulanic acid, which are antibiotics readily available on the Tanzanian market at the time of the study. The microdilution method was employed to determine beta-lactamase and ESBL production per the Clinical Laboratory and Standards Institute (CLSI) protocol. Cephalosporins, including ceftriaxone, cefuroxime and cefotaxime, the beta-lactamase inhibitor, and clavulanic acid, were serially diluted with concentrations ranging from 0.011 mg/ml to 200 mg/ml. Each of these antibiotics was subjected to sensitivity tests by determining the minimum inhibitory concentrations (MIC) of the clinical isolates of bacteria using a 96-well microdilution plate. Five microliters of bacterial suspension were inoculated into each well-containing 120µl of sterile Mueller-Hinton broth before incubation overnight. Results A total of 111 bacterial isolates were tested. Of the 111 tested bacterial isolates, 85 (76.6%) and 26 (23.4%) were Gram-negative and Gram-positive bacteria, respectively. Fifty-six clinical isolates (50.4%) were Escherichia coli, and 13 Salmonella species (11.7%) were among the Gram-negative isolates. On the other hand, 15 (13.5%) and 11 (9.9%) Gram-positive bacteria were Staphylococcus aureus and Streptococcus species, respectively, of all isolates. The majority of these clinical isolates, 71 (64.0%), were obtained from mid-stream urine, while the remaining were from stool, vaginal secretions, blood, pus, catheter sip, and urethra. A high proportion of tested Gram-negative bacteria, 58 (68.2%), were identified as ESBL producers, and 16 (61.5%) of the Gram-positive bacteria were identified as beta-lactamase producers. Cefuroxime was the least effective, exhibiting the largest MIC (18.47 ± 22.6 mg/ml) compared to clavulanic acid alone (5.28 ± 8.0 mg/ml) and clavulanic acid-cefuroxime (5.0± 12.32 mg/ml). Of all isolates, 78.2% were sensitive to chloramphenicol. Only five isolates had MIC larger than 32.23 mg/ml as opposed to cefotaxime and ampicillin, which had more isolates in that similar MIC range. Conclusion There is a high proportion of beta-lactamase, particularly ESBL-producing pathogens, in Dar es Salaam, Tanzania. Therefore, regular detection of beta-lactamase and ESBL production may help detect resistance to beta-lactam antibiotics.

Identification of GSK-3 as a Potential Therapeutic Entry Point for Epilepsy.

In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3'-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action.

Lipid Profile in HIV-Infected Patients Using First-Line Antiretroviral Drugs.

OBJECTIVE: To determine the prevalence of dyslipidemia in HIV-infected patients using the first-line antiretroviral (ARV) drugs triple regimen. METHODS: HIV-infected patients aged ≥15 years and attending Care and Treatment Clinic (CTC) at Muhimbili National Hospital, in Dar es Salaam, Tanzania, were recruited for the study. Blood samples from patients were collected during their regular visits at the CTC and assayed for determination of total cholesterol, triglycerides (TGs), high-density lipoprotein, low-density lipoprotein cholesterol, and CD4 counts. RESULTS: The median CD4 count was 346 cells/mm(3) (2-2600). Triple therapy combinations of ARV drugs used by patients included zidovudine (ZDV)/lamivudine (3TC)/efavirenz (EFV; 42.4%), ZDV/3TC/nevirapine (NVP; 33.8%), tenofovir (TDF)/emtricitabine/EFV (19.9%), and TDF/3TC/EFV (3.9%). The overall prevalence of dyslipidemia in patients was 77.5%. There were varied prevalence of derangement of individual lipids among patients. Age, body mass index, CD4 count, sex, and duration of ARV drug use were the predictors of poor lipid profiles. CONCLUSION: The findings of this study indicate the need for routine monitoring of lipids among HIV-infected patients.

Predictors of poor glycemic control in type 2 diabetic patients attending public hospitals in Dar es Salaam.

BACKGROUND: Tanzania has recently experienced a significant rise in the burden of diabetes, and it is estimated that more than 400,000 people are living with diabetes. A major concern in the management of diabetes is the occurrence of diabetic complications that occur as a result of poor glycemic control. Identification of the factors associated with poor glycemic control is important in order to institute appropriate interventions for the purpose of improving glycemic control and prevention of chronic complications. AIM: The aim of this study was to determine the level of glycemic control and explore the factors associated with poor glycemic control among patients with type 2 diabetes mellitus (T2DM). METHODOLOGY: A cross-sectional study was carried out at the diabetic clinics for T2DM patients at the national and municipal hospitals in Dar es Salaam. A total of 469 patients were enrolled over a period of 8 weeks from March 2013 to May 2013. Patients' information such as sociodemographic characteristics, self-care management behaviors, and medication adherence were obtained through interviews. Blood pressure, weight, and height were measured during the day of the interview. All available last readings for fasting blood glucose (FBG) measurements, lipid profile, and other clinical characteristics were obtained from patients' records. RESULTS: The mean age of patients was 54.93 years. The majority (63.5%) of patients were females and only eight patients had records of lipid profile measurements. Out of 469 patients, 69.7% had FBG of ≥7.2 mmol/L, indicating poor glycemic control. Females aged between 40 years and 59 years had significantly higher poor glycemic control (76.1%) as compared with their male counterparts. Thirty-eight percent of patients had poor medication adherence, which was associated with poor glycemic control. The proportion of poor glycemic control increased with age. A significantly high proportion of poor glycemic control was observed in patients who had had the disease for more than 20 years since diagnosis. Factors associated with poor glycemic control included lack of health insurance, using more than one oral hypoglycemic agent, normal body mass index, obesity, and nonadherence to diabetic medications. CONCLUSION: Patients in this study had generally poor glycemic control. From these findings it is recommended that diabetic patients should be routinely screened for lipid profile to determine levels of cholesterol, triglycerides, and low-density lipoproteins, which are risk factors for cardiovascular events. An education program should be developed to educate patients on the importance of medication adherence and weight management for better glycemic control.

Integration of Microfractionation, qNMR and zebrafish screening for the in vivo bioassay-guided isolation and quantitative bioactivity analysis of natural products.

Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation - generic for NP extracts of diverse origin - that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay-guided fractionation can be performed with only 20 mg of NP extract within a few days.

Zebrafish bioassay-guided natural product discovery: isolation of angiogenesis inhibitors from East African medicinal plants.

Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

Rational dispensing and use of artemether-lumefantrine during pregnancy in Dar es Salaam, Tanzania.

Artemether-Lumefantrine (ALu) is widely used for uncomplicated malaria during the second and third trimester of pregnancy. Because of the suspected teratogenic effects of artemether during the first trimester, quinine is used in early pregnancy unless the risks outweigh the benefits. The aim of this study was to assess dispensing practice of ALu in private pharmacies and knowledge of pregnant women regarding the use of ALu. This was a prospective-descriptive study involving visits to 200 private retail pharmacies (using a mystery shopper) and interviewing pregnant women at the municipal public hospitals in Dar es Salaam, Tanzania. Among the drug dispensers, 60 (30%) were pharmacists, 71(35.5%) nurse assistants, 34 (17%) pharmaceutical technicians and 35 (17.5%) sales persons with no formal education on drug dispensing. Among the dispensers, 14.5% had high knowledge, 38.0% had medium knowledge and 47.5% had low knowledge on the use of ALu during pregnancy. About thirty three percent of the drug dispensers were willing to dispense ALu during the first trimester of pregnancy. Sixty two percent of the drug dispensers indicated that ALu is the drug of choice for uncomplicated malaria after the first trimester of pregnancy. However, 36% indicated that ALu could not be used during pregnancy. A total of 200 pregnant women were interviewed. Among them, 16.5% were aware that ALu should not be taken during the first trimester of pregnancy. Only 17% of pregnant women were given information on the importance of taking food when using ALu, but none of them was given information on the importance of fatty meals when using ALu. In conclusion, the results show that most drug dispensers have inadequate knowledge about good dispensing practice of ALu in pregnancy. There is therefore a need for continuing training of drug dispensers regarding antimalarial drugs use in pregnancy.

Pattern of drug use among elderly patients in public and private hospitals in Dar-as Salaam.

OBJECTIVE: The aim of this study was to assess the use of potentially inappropriate medications in the elderly patients (> or = 65 years of age) according to the 2002 Beers criteria. METHOD: This was a retrospective and prospective study that was carried between December 2006 and June 2007. During this period, a total of 514 prescriptions of elderly patients were collected and examined for the pattern of drug use. The study involved reviewing prescriptions of patients who are 65 years old and above. Patients were those who were attending the clinics in the selected hospitals during the period of the study. In addition, files of patients who had attended the hospitals for not longer than the past 12 months were reviewed. The hospitals included two public hospitals (Mwananyamala municipal hospital and Muhimbili National Hospital) and two privately-owned hospitals (Aga Khan and Hindu Mandal hospitals) in Dar es Salaam. RESULTS: The study revealed that 11.4% of medicines prescribed were drugs of concern i.e. medications that should be avoided in elderly patients or which are inappropriate for use in elderly patients according to Beers list. Among these drugs those with high frequency of use were Nifedipine (3.5%), Iron sulphate (2.2%), Chlorpropamide (2%) and Digoxin (1.8%). These drugs were more prescribed in the public hospitals than in private ones. It was also observed that more women (62.3%) are suffering from a number of diseases than males (37.7%). In addition, women received more inappropriate medications (60.7%) than their male counterparts (393%). The common most prevalent diseases were hypertension (22.5%), diabetes mellitus (17.9%), malaria (13.5%), urinary tract infection (4.2%) and anemia (3.7%). CONCLUSION: Based on the findings, there is a need for both the public and health care professionals to be aware of the harmful drugs in the old age so as to minimize occurrence of side effects.

In vitro accumulation and permeation of hypericin and lipophilic analogues in 2-D and 3-D cellular systems.

Previous studies have shown that hypericin is an excellent diagnostic tool for the fluorescence detection of carcinoma in situ in the human bladder. The present work was performed to get a better insight into the mechanism of cellular uptake of hypericin (HYP) using RT-112 human papillary TCC cells of the bladder. Using lipophilic hypericin acid amide derivatives like hypericin acid hexylamide (AM6), hypericin acid octylamide (AM8) and hypericin acid dodecylamide (AM12), the effect of increased lipophilicity on the binding to serum proteins was investigated, as well as the cellular accumulation and permeation, both in 2-D and 3-D cell conditions. Density-gradient ultracentrifugation of the compounds pre-incubated with fetal bovine serum (FBS) showed that HYP and to a lesser extent AM6 predominantly bind to LDL, whereas AM8 and especially AM12 preferably associate with HDL. The cellular accumulation of the compounds did not significantly differ when LDL or AcLDL was supplemented to medium, and with all compounds the highest uptake could be observed in case of medium without supplements. Using medium without supplements it was further observed that the compounds with the highest lipophilicity accumulated substantially less in RT-112 cells. We further found a significant difference in the intracellular concentration of AM8 and AM12 when LDL or FBS was supplemented to MEM, but not in case of HYP and AM6. Of particular interest, AM8 and especially AM12 showed enhanced intraspheroidal permeation in the presence of FBS. It is believed that the relative stronger binding to HDL reduces the intracellular accumulation, as seen in the 2-D conditions, and therefore increases the probability of paracellular transport in a 3-D multicellular system by passive diffusion. In conclusion the data suggest that the amount of free hypericin or its lipophilic congener determines the extent of intracellular accumulation. This concentration is both determined and limited by binding to different lipoproteins present in the medium, and by the formation of stable homoaggregates. The findings further highlight AM8 and AM12 as compounds better tailored for paracellular transport than HYP itself and therefore as potentially very interesting diagnostic tools for TCC lesions in the bladder.

Enhancing the photodynamic effect of hypericin in human bladder transitional cell carcinoma spheroids by the use of the oxygen carrier, perfluorodecalin.

In the present study, we evaluated the possibility of enhancing the photodynamic effect of hypericin in transitional cell carcinoma (TCC) spheroids by the use of the oxygen carrier, perfluorodecalin. Following incubation with hypericin, RT-112 TCC spheroids were irradiated in the presence or absence of perfluorodecalin, at light doses of 7 J/cm(2) or 28 J/cm(2) , delivered at a fluence rate of 15 mW/cm(2) . The photodynamic therapy (PDT) efficacy was evaluated and apoptotic cells were visualized. The results show that, in the absence of perfluorodecalin, spheroidal TCC cells are inadequately sensitive to hypericin PDT. As was shown by fluorescence microscopy, this lack of activity was not due to insufficient photoactive concentrations of hypericin reaching the inner parts of the spheroids. Conversely, enhanced oxygenation of spheroids by perfluorodecalin led to a dramatic enhancement of hypericin PDT efficacy. The detection of nuclear shrinkage or fragmentation with DAPI staining and the assessment of cell morphology by light microscopy indicated that apoptosis was the most prominent response of spheroids to hypericin PDT in the presence of perfluorodecalin. In conclusion, the results of this study suggest that perfluorocarbons, such as perfluorodecalin, are useful in enhancing the oxygenation of tumor tissue, resulting in highly efficient hypericin PDT. Since hypericin becomes concentrated specifically in human bladder urothelial carcinoma lesions and the bladder is very well suited to instillation with a perfluorocarbon, combining the techniques looks very promising for an efficient and selective whole bladder wall photodynamic antitumoral treatment in a urological clinical setting.

Permeation of hypericin in spheroids composed of different grade transitional cell carcinoma cell lines and normal human urothelial cells.

PURPOSE: We investigated the importance of E-cadherin expression on the selective accumulation of hypericin in superficial bladder cancer after intravesical instillation. MATERIALS AND METHODS: Spheroids obtained from a panel of 3 transitional cell carcinoma cell lines, namely J-82, RT-4 (American Type Culture Collection, Manassas, Virginia) and RT-112 (German Collection of Micro-organisms and Cell Cultures, Braunschweig, Germany), and normal human urothelial (NHU) cells were incubated with hypericin. Accumulation was examined with fluorescence microscopy. Immunohistochemical staining was used to assess E-cadherin expression. RESULTS: Immunohistochemical staining showed E-cadherin expression in NHU (++), RT-112 (+) and RT-4 (+) spheroids, whereas E-cadherin expression was absent in J-82 spheroids. The highest intraspheroidal hypericin accumulation was observed in transitional cell carcinoma spheroids, whereas limited permeation was seen in NHU spheroids. Taken together the data point to an inverse relationship between E-cadherin expression and the permeation of hypericin throughout a 3-dimensional cellular matrix. CONCLUSIONS: Loss of E-cadherin expression correlates with loss of intercellular adhesion, tight junction formation and enhanced paracellular transport. The data show that E-cadherin hampers the permeation of hypericin in spheroids and the loss of intercellular adhesion, present in superficial bladder cancer lesions, can be associated with enhanced hypericin permeation. Therefore, E-cadherin expression seems to have a pivotal role in the selective uptake of hypericin after intravesical instillation in human bladders.

Enhancing the photodynamic effect of hypericin in tumour spheroids by fractionated light delivery in combination with hyperoxygenation.

The aim of this study was to explore the hypothesis of oxygen depletion during light irradiation as a possible explanation for the incomplete response seen after hypericin-mediated photodynamic therapy (PDT) under specific conditions. To investigate this, we performed PDT experiments using transitional cell carcinoma spheroids with fractionated light irradiation and hyperoxygenation. After 2-h incubation with 3 different hypericin concentrations, spheroids were irradiated either continuously or with fractionated light delivery. The effect of hyperoxygenation was investigated by bubbling normobaric oxygen in the solution surrounding the spheroids before continuous irradiation or during the dark interval of light fractionation. The PDT efficacy was evaluated with an MTT antiproliferation assay and apoptotic cells were visualized after PDT by DAPI staining. Our results show that fractionated light delivery with dark intervals ranging from 1 to 10 min does not enhance the PDT efficacy in spheroids at all, whereas hyperoxygenation, using appropriate hypericin concentrations and oxygenation intervals, results in a virtually complete malignant cell killing through apoptosis. This study suggests that oxygen depletion is the major source of relative treatment failure in hypericin-mediated PDT with spheroids, which can only be overcome with hyperoxygenation. Therefore, whole bladder wall PDT with hypericin is likely to become a very efficient antitumoural treatment against superficial bladder cancer, on the condition that instillation fluids are hyperoxygenated during light irradiation.

Stability of different formulations and ion pairs of hypericin.

Hypericin, solubilized in an instillation fluid consisting of an aqueous buffer supplemented with 1% plasma proteins, is currently used as a clinical diagnostic tool for the detection of superficial TCC (transitional cell carcinoma) tumors. However, the development of a sterile and stable hypericin stock formulation, excluding the presence of plasma constituents, would be an important factor in a more general clinical application of the method. Therefore, we investigated the stability of several heat sterilized hypericin formulations and ion pairs. Besides sodium hypericinate (in distilled water, in phosphate buffer, in polyethyleneglycol (PEG) 400), several other hypericinate salts (potassium, lysine, TRIS or hexylamine) were investigated. As to that, the physical appearance of different hypericin concentrates stored at 4 and 37 degrees C was investigated. Besides, after dilution into cell culture medium, the ability of hypericin remaining to accumulate in tumor cells and demonstrating photocytotoxic effects upon light irradiation was assessed. These findings suggest that PEG 400 is an excellent hypericin formulation, since it maintained the stability of the compound for at least 120 d when stored at either 4 or 37 degrees C. PEG 400 therefore is a suitable vehicle for the storage of hypericin prior to preparation of the bladder instillation solution.

In vivo accumulation of different hypericin ion pairs in the urothelium of the rat bladder.

OBJECTIVE: To optimise the diagnostic and phototherapeutic efficacy of hypericin in superficial bladder cancer, by developing a bladder instillation fluid that does not depend on the presence of plasma proteins for an appropriate and reliable urothelial uptake of hypericin. MATERIALS AND METHODS: Sodium hypericinate (in distilled water, in sodium phosphate buffer, or in polyethylene glycol) and several other hypericinate salts (potassium, lysine, TRIS or hexylamine) were instilled with no plasma constituents into the rat bladder. The accumulation of hypericin was assessed with fluorescence microscopy. RESULTS: The diagnostic and phototherapeutic efficacy of hypericin depends on its ability to penetrate the tumour lesions sufficiently to show a fluorescent signal or elicit a photodynamic response. Several instillation fluids meet the purpose, as the urothelial accumulation of hypericin was similar to that obtained with the instillation fluid supplemented with plasma proteins, used in clinical practice. The highest concentrations of hypericin in the urothelium of the rat bladder were obtained with hypericin instillation solutions prepared with distilled water or 20% polyethylene glycol 400 in distilled water. Fluorescence microscopy showed that hypericin was selectively localized in the urothelium. Furthermore, all variables investigated (hydrophilic/lipophilic balance, pH, saline, presence of organic solvent) can dramatically influence the in vivo accumulation of hypericin. CONCLUSION: An appropriate and reliable urothelial uptake of hypericin does not depend on the presence of plasma protein supplements in the bladder instillation fluid.

Accumulation and photocytotoxicity of hypericin and analogs in two- and three-dimensional cultures of transitional cell carcinoma cells.

The aim of this study was to investigate the in vitro cellular accumulation, distribution and photocytotoxic effect of hypericin in two-dimensional (2-D) and three-dimensional (3-D) cultured RT-112 transitional cell carcinoma cells of the bladder. In addition, two iodinated derivatives of hypericin were incorporated to investigate whether these analogs, with their increased lipophilicity and heavy-atom effect, display a different biological behavior and optimized photodynamic effect. The results indicate that hypericin and mono-iodohypericin behave similarly in terms of cellular accumulation, spheroidal distribution and photocytotoxic effect. In contrast, di-iodohypericin concentrated to a higher extent in monolayers and spheroids, but the accumulation was restricted to the outermost part of the spheroid. An inverse correlation therefore seems to exist between the extent of cellular uptake under 2-D conditions and the penetration of the compounds in multicellular systems. Moreover, a less pronounced photocytotoxic effect was observed for di-iodohypericin in both 2-D and 3-D cell culture systems. It can be concluded that iodinated derivatives of hypericin do not show an increased cytotoxic effect upon irradiation in either monolayers or spheroids. Moreover, this study shows that when new photosensitizers are preclinically developed, the use of 3-D cell aggregates is critical for a correct evaluation of their efficacy.