Yield of family screening in dilated cardiomyopathy within low-income setting: Tanzanian experience.

BACKGROUND: Dilated cardiomyopathy (DCM) is often familial and screening of relatives is recommended. However, studies on the yield of screening are scarce in developing countries. AIM: The aim of the study was to identify and characterise First-degree relatives of patients with DCM in Tanzania. METHODS: We recruited first-degree relatives of 57 DCM patients. DCM in the relatives was diagnosed using the 2016 revised definition by the European Society of Cardiology working group on myocardial and pericardial diseases. RESULTS: We screened 120 first-degree relatives. All were asymptomatic (100%) with a median age of 39.0 years (29.5-49.0), slightly over a half (53.3%) were females and 17 (14.1%) were found to have previously unknown DCM. The mean (± SD) indexed left ventricular end-diastolic volume was significantly higher in relatives with DCM (71 ± 11.5 ml) compared to relatives without DCM (50 ± 11.5) (p = 0.001). CONCLUSION: First-degree relatives of patients with DCM are at risk of developing asymptomatic DCM at a young age.

Risk Factors and Prevalence of Dilated Cardiomyopathy in Sub-Saharan Africa: A Systematic Review.

Highlights  Prevalence of DCM varies widely in SSA.Cardiovascular risk factors are important in patients with DCM.The role of genetics in idiopathic DCM is not studied in major part of SSA.

CYP2B6*6 genotype and high efavirenz plasma concentration but not nevirapine are associated with low lumefantrine plasma exposure and poor treatment response in HIV-malaria-coinfected patients.

We investigated the influence of efavirenz (EFV)- or nevirapine (NVP)-based antiretroviral therapy (ART) on lumefantrine plasma exposure in HIV-malaria-coinfected patients and implication of pharmacogenetic variations. A total of 269 HIV patients with uncomplicated falciparum malaria on NVP-based ART (NVP-arm), EFV-based ART (EFV-arm) or not receiving ART (control-arm) were enrolled and treated with artemether-lumefantrine. Day-7 lumefantrine, baseline EFV and NVP plasma concentrations, and CYP2B6*6,*18, CYP3A4*1B, CYP3A5*3,*6,*7, ABCB1 c.3435C>T and ABCB1 c.4036A>G genotypes were determined. The median day-7 lumefantrine plasma concentration was significantly lower in the EFV-arm compared with that in NVP- and control-arm. High EFV plasma concentrations and CYP2B6*6/*6 genotype significantly correlated with low lumefantrine plasma concentrations and high rate of recurrent parasitemia. No significant effect of NVP-based ART on lumefantrine exposure was observed. In conclusion, owing to long-term CYP3A induction, EFV-based ART cotreatment significantly reduces lumefantrine plasma exposure leading to poor malaria treatment response, which is more pronounced in CYP2B6 slow metabolizers.

Dispensing Practice of Prescribed Medicines in the Private Pharmacies in Urban Areas of Tanzania.

A descriptive cross-sectional study was conducted to assess quality of dispensing and knowledge of dispensers in 206 private retail pharmacies. The study was conducted in Dar es Salaam, Tanzania between September 2011 and April 2012. Patient simulation (mystery shopper) approach was used to assess dispensing skills of drug dispensers for prescription only medicines. In assessing dispensing skills, a 7-days course of metronidazole tablets was bought from each pharmacy. The knowledge of drug dispenser's regarding dispensing of prescription only medicines was assessed through focus group discussions and interviews. Majority (70.4%) of drug dispensers were not trained pharmaceutical personnel. The level of dispensing skills ranged from low (25.7%) to medium (70.4%). Majority of drug dispensers had low (11.4%) to medium (83.2%) levels of knowledge about dispensing of 'prescription only' medicines. From these findings, it is recommended that the national Pharmacy Council should ensure that prescription only medicines are dispensed by trained pharmaceutical personnel. On job training and continuing professional development should also be emphasized to build capacity of drug dispensers.

Rational use and effectiveness of morphine in the palliative care of cancer patients at the Ocean Road Cancer Institute in Dar es Salaam, Tanzania.

Morphine and other opioids is the mainstay of cancer pain management. However, considerable fears surrounding their use present barriers to pain control. The aim of this study was to assess the rational use and effectiveness of morphine for management of pain in the palliative care of cancer patients at Ocean Road Cancer Institute (ORCI) in Tanzania. A total of 100 cancer patients who were receiving morphine therapy at the ORCI were interviewed to get information on morphine use. In addition, information on the prescribed doses of morphine was obtained from medical records of 200 patients who have used morphine from September 2005 to April 2006. Both outpatients and inpatients with advanced cancer who were receiving morphine for palliative care were involved. Seven (7) palliative caregivers, including two doctors, two nurses, a pharmacist, a pharmaceutical technician and a social worker were also interviewed. Of the 100 interviewees, 37% were aware of morphine. The level of education and duration of therapy had an impact on the awareness. The results also showed that oral morphine solution was the most common route (96%) of administration. Fifty-seven percent of the patients described the doses of morphine given to be effective in relieving their pain. Although most patients (79%) experienced morphine-induced side effects, the majority (93%) were continuing with the therapy. There were no indication of irrational use of morphine and morphine-induced side effects were well managed. The majority of patients and caregivers had positive attitude towards the use of morphine. In conclusion, the study revealed that the use of morphine is acceptable among a large proportion of patients receiving palliative care and that the majority of them find the doses given effective to relieve their pain.

Investigation of fractions present in the stem bark of Annickia kummeriae on their P-glycoprotein inhibitory pump activity.

Using MCF-7R cells and rhodamine 6G as the fluorescent probe, a bioassay-targeted purification process was pursued in order to isolate the active P-gp inhibitory fractions from Annickia kummeriae. Of 24 fractions obtained in the first preparative liquid chromatography (p-LC) run, only fraction 1 exhibited activity. Further p-LC fractionation led to the separation of fraction 1 into fractions 1.1-1.8. Fractions 1.4, 1.5 and 1.6 proved to be active by inducing a significant accumulation of rhodamine 6G by 3.3-, 4.5- and 4.9-fold at 10 microg/mL, and by 5.3-, 6.3- and 6.8-fold at 100 microg/mL, respectively. Fraction 1.6 was separated into several fractions by using an analytical liquid chromatography (a-LC) system. Fractions 1.6.18, 1.6.19 and 1.6.20 were active and they induced an accumulation of rhodamine 6G by 3.0-, 1.8- and 3.5-fold at 1x microg/mL and by 4.8-, 6.7- and 6.8-fold at 10x microg/mL, respectively. Afterwards, 28.3 mg of fraction 1.6 was processed by a-LC, and fractions 1.6.18, 1.6.19 and 1.6.20 were collected separately and dried. The amounts of materials recovered were 6.2, 7.4 and <1 mg, corresponding to 21.9%, 26.1% and <3.5% of fraction 1.6, respectively. From the total amount injected and the relative masses represented by these fractions, it can be calculated that the 1x microg/mL level corresponded to ca. 35, 42 and <5 microg/mL, respectively. Fluorescence microscopy revealed that incubation of the cells with rhodamine 6G alone did not show any fluorescence, whereas cells which were incubated in medium containing rhodamine 6G together with fraction 1.4, 1.6 or reserpine, clearly indicated accumulation of the dye intracellularly. This is an indication that the active compounds effected high intracellular fluorescence by inducing accumulation of the dye in the cells through inhibition of the P-gp pump.

Screening of Tanzanian plant extracts for their potential inhibitory effect on P-glycoprotein mediated efflux.

For years, many efforts have been made to discover new drugs using plants as natural screening libraries. In this study, extracts of 43 Tanzanian medicinal plants were screened for their potential inhibitory effect on P-gp, using the secretory transport of Cyclosporin A (CsA) in the Caco-2 system as a measure of the functionality of P-gp efflux. Two out of these 43 plant extracts (extracts of Annickia kummeriae and Acacia nilotica) appeared to have a modulatory effect on P-gp related efflux carriers. In presence of the extract of Annickia kummeriae, a concentration dependent decrease on the polarity in transport of CsA was observed; the inhibitory effect of this extract on P-gp was comparable to that of valspodar, a known P-gp inhibiting agent. The exact nature of the active components of these botanicals remains to be identified.

Hypericin-based fluorescence diagnosis of bladder carcinoma.

OBJECTIVE: To determine the use of hypericin instillation for the fluorescent detection of papillary bladder cancer and carcinoma in situ. PATIENTS AND METHODS: Eighty-seven patients with papillary bladder cancer and/or carcinoma in situ received instillations with 40 mL of an 8 micromol/L hypericin solution for at least 2 h. Fluorescent excitation with blue light was effective for up to 16 h, and biopsies were examined by fluorescence microscopy. RESULTS: There were no side-effects reported, no photobleaching and all papillary lesions fluoresced red. The sensitivity and specificity for detecting carcinoma in situ was 94% and 95%, respectively. An interval of 4 months is recommended after BCG instillations before using this test. Fluorescence microscopy showed that hypericin was selectively localized in the epithelium. CONCLUSIONS: Hypericin-induced fluorescence has a high sensitivity and specificity for detecting bladder cancer. After 4 months there are few false-positive results in patients treated with BCG.

Cellular photodestruction induced by hypericin in AY-27 rat bladder carcinoma cells.

In a recent clinical study we showed that hypericin accumulates selectively in urothelial lesions following intravesical administration of the compound to patients. In the present study the efficacy of hypericin as a photochemotherapeutic tool against urinary bladder carcinoma was investigated using the AY-27 cells (chemically induced rat bladder carcinoma cells). The uptake of hypericin by the cells increased by prolonging the incubation time and increasing the extracellular hypericin concentration. Photodynamic treatment of the cells incubated with 0.8 and 1.6 microM hypericin concentrations resulted in remarkable cytotoxic effects the extent of which depended on the fluence rates. Photoactivation of 1.6 microM hypericin by 0.5, 1.0 or 2.0 mW/cm2 for 15 min resulted in 3, 30 and 95% of the antiproliferative effect, respectively. Increasing the photoactivating light dose from 0.45 to 3.6 J/cm2 resulted in a five-fold increase in hypericin photodynamic activity. Irrespective of the fluence rates and irradiation times incubation of the cells with 10 microM hypericin induced rapid and extensive cell death in all conditions. The type of cell death (apoptosis or necrosis) induced by photoactivated hypericin depended largely on the hypericin concentration and the postirradiation time. At lower hypericin concentrations and shorter postirradiation times apoptosis was the prominent mode of cell death; increasing the hypericin concentration and/or prolonging the postirradiation time resulted in increased necrotic cell death. Cell pretreatment with the singlet oxygen quencher histidine, but not with the free-radical quenchers, significantly protected the cells from photoactivated hypericin-induced apoptosis, at least when a relatively low concentration (1.25 microM) was used. This result suggests the involvement of a Type-II photosensitization process. However, cells treated with higher hypericin concentrations (2.5-5 microM) were inadequately protected by histidine. Since hypericin is thus shown to be a potent and efficient photosensitizer, and since the conditions used were the same as when hypericin is used clinically to locate early-stage urothelial carcinoma lesions, hypericin may well become very important for the photodynamic treatment of superficial bladder carcinoma.

In vivo photodynamic activity of hypericin in transitional cell carcinoma bladder tumors.

In a recent clinical study, we showed that hypericin accumulates selectively in urothelial lesions of the bladder following intravesical administration of the compound in patients. This observation infers that hypericin, a potent photosensitizer, could be used as a selective photodynamic therapy (PDT) tool against superficial bladder cancer. In the present study we investigated the in vivo PDT activity of hypericin in transition cell carcinoma (TCC) tumors of the bladder. Both the distribution and tumor PDT response were carried out using subcutaneous heterotopic AY-27 TCC tumors in syngeneic rats. For both PDT and distribution studies, hypericin (1 or 5 mg/kg) was injected intravenously 0.5, 6 or 24 h before PDT or distribution evaluation. The data show that hypericin is a potent photosensitizer in the treatment of TCC tumors in vivo and that the interval between drug administration and photo-irradiation has a dramatic effect on the PDT outcome. Using a 0.5 h interval between drug administration and photo-irradiation the tumor regrowth study indicated that no tumor mass could me measured 9-10 days after PDT. On the contrary, lengthening the time interval between drug administration and photo-irradiation resulted in a gradual loss of PDT efficiency in these tumors. For instance, while the 6 h drug interval protocol produced a moderate PDT activity in which the tumor sizes decreased to about 50% of their original sizes 11-16 days after photo-irradiation, the 24 h interval protocol was even less effective. The distribution data indicate that the PDT efficiency of hypericin in TCC tumors corresponded to the plasma concentrations rather than to the over all concentrations in the tumor. It is therefore conceivable that the mechanism of PDT efficacy of hypericin in TCC tumors is through indirect (vascular effects) rather than through direct effects (cellular destruction) of hypericin in these tumors. In conclusion, our data indicate that hypericin is a potent photosensitizer against AY-27 TCC tumors and that the PDT efficacy of hypericin is largely determined by photosensitizer distribution in the tumor at the time of photo-irradiation.

Photodynamic activity of hypericin in human urinary bladder carcinoma cells.

Recently, we reported the selective accumulation of hypericin in transitional cell carcinoma cells following intravesical instillation of hypericin in humans. This observation infers that hypericin, a potent photosensitizer, could be used as a selective PDT (photodynamic therapy) tool against superficial bladder cancer. The aim of the present study was to investigate in vitro whether hypericin exhibits specific affinity for TCC transitional cell carcinoma) bladder cells and to assess its photocytotoxic effect. Three human TCC cell lines (J-82, T-24 and RT-4), a chemically induced rat TCC cell line (NBT-II), but also non-bladder carcinoma cells (HeLa, A431, MCF-7 and MCF-***ADR) and normal cells (HEL229, RPE and PHK), were used in this comparative study. Flow cytometric analysis of cells treated with different hypericin-containing vehicles for various incubation times (2 hours or 24 hours) indicated that short exposure of the cells (2 hours) to hypericin in the absence of serum results in the highest intracellular accumulation of the compound. As expected, prolonging the incubation time increased both the cellular accumulation and photocytoxicity of hypericia. With the exception of the RT-4 and MCF-7 cells (which were less sensitive to hypericin), all the other carcinoma cell lines examined showed equal sensitivity to the photoactivated hypericia, independently of their histological origin (bladder or non-bladder). Moreover, normal cells exhibited the same pattern of hypericin photosensitivity as shown by the cancer cells, indicating that, in cultured cells, hypericin cellular uptake and subsequent photokilling is not selective. This suggests that in vivo factors other than the cancer cells themselves are responsible for the specific accumulation of hypericin in urothelial carcinoma lesions.

A comparative study of the photosensitizing characteristics of some cyanine dyes.

The present work has been carried out to explore the potential application of cyanines in photodynamic therapy. After photosensitization, the in vitro cytotoxic and antiproliferative activity on HeLa cells of a total of 35 cyanines belonging to several chemical subgroups is explored. Most of these cyanines have never been used before in similar experimental work. From a first set of experiments, it is found that none of the krypto-, oxa- and imidacyanines is photobiologically active on HeLa cells. Conversely, five thiacyanines (Thiacl-5), one rhodacyanine (Rhodac) and four indocyanines (Indoc2, Indoc4, Indoc5, Indoc7) show photodependent cytotoxicity or antiproliferative effects. A more detailed study shows that out of the ten selected compounds, eight cyanines feature significant photodependent cytotoxic and antiproliferative effects. All possess maximum absorption ranges between 545 and 824 nm. In particular, Rhodac, a tetramethinemeromonomethine rhodacyanine dye with an absorption maximum of 655 nm (ethanol) and a molar absorption coefficient epsilon = 108000 shows very promising photo-dependent biological activity. In general, the measured singlet oxygen quantum yield of the selected cyanines is low (< 0.08) and does not correlate with the degree of photosensitization. Furthermore, the present study shows that cyanines with a partition coefficient close to 1.5 accumulate to the highest extent in HeLa cells, while the more hydrophobic compounds (e.g., indocyanines) concentrate less intracellularly.

Investigation of the absorption of hypericin into the skin of hairless mice.

The skin absorption of hypericin was evaluated in hairless mice to develop an optimised hypericin topical formulation that could be used in the clinical study of psoriasis. Hypericin (0.01-1.0%) in Beeler basis, polyethylene glycol ointment, carbopol gel, cetomacrogol cream, petrolatum or emulsifying ointment, with and without skin-absorption enhancers (isopropylidene glycerol and diethylene glycol monoethyl ether), was tested in-vivo on hairless mice skin. Using a skin-stripping technique and the intrinsic fluorescence of hypericin under standardised UV365 irradiation, it was demonstrated that the absorption of hypericin very much depended on the vehicle used. The concentrations of hypericin in the skin were then estimated by HPLC analysis. For this purpose, two vehicles were employed, with which hypericin penetrated the skin of hairless mice well (emulsifying ointment with isopropylidene glycerol) or very poorly (polyethylene glycol ointment). In the case of emulsifying ointment with isopropylidene glycerol (0.05% hypericin), a substantial concentration of hypericin (8.6+/-3.2 microg g(-1)) (mean +/- s.d., n = 5) was found in the skin. With polyethylene glycol ointment, however, only a limited hypericin skin concentration (0.38+/-0-34 microg g(-1), n = 5) was achieved. These results show that emulsifying ointment with polyethylene glycol holds promise as an effective topical vehicle for the treatment of skin diseases, such as psoriasis, with hypericin.

Cytotoxicity of some medicinal plant extracts used in Tanzanian traditional medicine.

Using the ethnomedical data approach, some Tanzanian plants that are used in Tanzanian traditional medicine for cancer or non-cancer diseases were collected and evaluated for cytotoxic activity. The antiproliferative effect of the methanolic extracts (10 and 100 microg/ml) of 47 plants was evaluated in vitro on three human cell lines (HeLa, cervical carcinoma; HT29, colon adenocarcinoma; and A431, skin carcinoma). From the nine plants that are used to treat cancer, two plants (22%) exhibited pronounced cytotoxic effect (<25% cell proliferation) at least in one of the tested cell lines. For the 38 plants that are used to treat non-cancer diseases, 14 plants (37%) exhibited pronounced cytotoxic effect (<25% cell proliferation). Cell type cytotoxic specificity was observed in some extracts. Overall, the A431 cells were much more sensitive to most of the extracts than the other cell lines. For the plants that are used as anticancer herbal drugs, our results indicate that there is no correlation between the reported use of these plants and their cytotoxic activity obtained in this study. However, plants that have shown pronounced cytotoxic activity will be evaluated further for the possible isolation of active antitumor compounds.

Evidence that total extract of Hypericum perforatum affects exploratory behavior and exerts anxiolytic effects in rats.

Clinical trials have extensively reported the ability of Hypericum perforatum extracts to exert a significant antidepressant activity. Hypericin, the main constituent of H. perforatum extract, is no more regarded as the active principle of the antidepressant activity of the drug. Hence, the question of which constituents are involved in the basic activity of the total extract, is still waiting for an answer. In the present study we focused our attention on the potential anxiolytic activity of H. perforatum total extract, and of some pure components such as protohypericin and a fraction containing hypericin and pseudohypericin. Herein we report that the total extract of H. perforatum increases the locomotor activity in the open field and exerts anxiolytic activity in the light-dark test, whereas the single components did not show any effect. Interestingly, the anxiolytic activity of the total extract was blocked by pretreatment of rats with the benzodiazepine antagonist Flumazenil, hence suggesting an implication of benzodiazepine receptor activation in the anxiolytic effect of H. perforatum extract. Electrophysiological studies, performed to gain more information on the mechanism of action, showed that hypericin reduced the GABA-activated chloride currents, while pseudohypericin did an opposite effect. Furthermore, both hypericin and pseudohypericin inhibited the activation of NMDA receptors.

In vitro transport and uptake of protohypericin and hypericin in the Caco-2 model.

The intestinal absorption characteristics of protohypericin, a protonaphthodianthrone present in Hypericum extract, were studied and compared with those of hypericin. The Caco-2 model was used as a model of the intestinal mucosa to assess transepithelial transport and cell uptake. The experimental work was performed in specific light conditions that prevented both the photoconversion of protohypericin into hypericin and the photosensitization of the cells. Following application of the individual compounds (80-200 microM) to the apical side of the monolayers, the appearance in the basolateral compartment was found to be very low (<0.5%/5 h), but was comparable for both compounds. A lag-time of 2-3 h was observed, suggesting gradual saturation of binding sites on the membrane or inside the cells. Uptake experiments of protohypericin and hypericin by Caco-2 cells revealed a very significant cellular accumulation (4-8%); uptake was characterised by saturation after 3 h. The findings of this study suggest that protohypericin has comparable absorption characteristics as hypericin and may contribute to the beneficial effect of Hypericum extract after oral dosing.

Skin photosensitization with topical hypericin in hairless mice.

Hypericin, a naturally occurring photosensitizer, exhibits interesting in vitro photobiological activities, which suggest that the compound is a potential antipsoriatic agent. In this study, the possibility of hypericin penetrating the skin in photo-active concentrations has been studied. Hypericin is incorporated in either emulsifying ointment supplemented with solketal (hypericin content: 0.05%) or in polyethylene glycol (PEG) ointment (hypericin content: 0.5%) and applied to the skin of hairless mice for 4 h. After removing excess ointment, the mice are then irradiated with different light doses using a 500 W halogen lamp. As a positive control, intraperitoneally (i.p.) administered hypericin (10 and 40 mg/kg) has also been tested. Erythema, desquamation and erosions are demonstrated in the mice treated with hypericin in emulsifying ointment with solketal using a light dose of at least 4.5 J/cm2. In general, these reactions correlate well with those of i.p. administered hypericin (40 mg/kg), indicating that hypericin incorporated in emulsifying ointment with solketal is well absorbed by the skin of the mice. However, for the i.p. administered hypericin (40 mg/kg), we could not evaluate phototoxic reactions in the group of animals that received a light dose of 108 J/cm2, as they all died 12-24 h after irradiation, indicating extreme photosensitization with systemic hypericin at higher light doses. On the contrary, there is no measurable skin photosensitivity induced by hypericin when incorporated in PEG ointment or when 10 mg/kg hypericin is i.p. administered. Our results show that hypericin incorporated in a suitable vehicle can be delivered to the skin in photo-active concentrations. Using a vehicle such as emulsifying ointment with solketal, it will be possible to explore the photo-activity of hypericin in the treatment of psoriasis and other skin diseases.

Photocytotoxicity of protohypericin after photoconversion to hypericin.

In the present study, protohypericin was synthesised in order to compare its intrinsic photocytotoxicity with that of hypericin. The experimental work was performed in specific filtered light conditions that prevented both an unintended photoconversion of protohypericin and photosensitization of the cells. Assessing the photocytotoxicity as a function of irradiation time, it was found that the photocytotoxicity of both compounds converged after a long irradiation time (i.e., 15 min), while the difference between the photocytotoxicities was maximal after a short irradiation time (i.e., 1 min). Since this could not be accounted for by a redistribution of protohypericin during irradiation, and the different irradiation times corresponded to different degrees of photoconversion of protohypericin into hypericin, the results clearly suggest that protohypericin exhibits intrinsically a dramatically lower photoactivity as compared to hypericin.

Photocytotoxic effect of pseudohypericin versus hypericin.

Pseudohypericin and hypericin, the major photosensitizing constituents of Hypericum perforatum, are believed to cause hypericism. Since hypericin has been proposed as a photosensitizer for photodynamic cancer therapy, the photocytotoxicity of its congener pseudohypericin has been investigated. The presence of foetal calf serum (FCS) or albumin extensively inhibits the photocytotoxic effect of pseudohypericin against A431 tumour cells, and is associated with a large decrease in cellular uptake of the compound. These results suggest that pseudohypericin, in contrast to hypericin, interacts strongly with constituents of FCS, lowering its interaction with cells. Since pseudohypericin is two to three times more abundant in Hypericum than hypericin and the bioavailabilities of pseudohypericin and hypericin after oral administration are similar, these results suggest that hypericin, and not pseudohypericin, is likely to be the constituent responsible for hypericism. Moreover, the dramatic decrease of photosensitizing activity of pseudohypericin in the presence of serum may restrict its applicability in clinical situations.