Mechanistic Modeling of Wet Stirred Media Milling for Production of Drug Nanosuspensions.

Drug nanocrystals have been used for a wide range of drug delivery platforms in the pharmaceutical industry, especially for bioavailability enhancement of poorly water-soluble drugs. Wet stirred media milling (WSMM) is the most widely used process for producing dense, stable suspensions of drug nanoparticles, also referred to as nanosuspensions. Despite a plethora of review papers on the production and applications of drug nanosuspensions, modeling of WSMM has not been thoroughly covered in any review paper before. The aim of this review paper is to briefly expose the pharmaceutical scientists and engineers to various modeling approaches, mostly mechanistic, including computational fluid dynamics (CFD), discrete element method (DEM), population balance modeling (PBM), coupled methods, the stress intensity-number model (SI-SN model), and the microhydrodynamic (MHD) model with a main focus on the MHD model for studying the WSMM process. A total of 71 studies, 30 on drugs and 41 on other materials, were reviewed. Analysis of the pharmaceutics literature reveals that WSMM modeling is largely based on empirical, statistically based modeling approaches, and mechanistic modeling could help pharmaceutical engineers develop a fundamental process understanding. After a review of the salient features and various pros-cons of each modeling approach, recent advances in microhydrodynamic modeling and process insights gained therefrom were highlighted. The SI-SN and MHD models were analyzed and critiqued objectively. Finally, the review points out potential research directions such as more mechanistic and accurate CFD-DEM-PBM simulations and the coupling of the MHD-PBM models with the CFD.

Candesartan protects from cisplatin-induced kidney damage via the GDF-15 pathway.

OBJECTIVE: The aim of this study was to explore the protective effect of candesartan against cisplatin-induced kidney damage, with a specific focus on the growth differentiation factor 15 (GDF-15) pathway. MATERIALS AND METHODS: 24 adult female Wistar rats, with a weight range of 200-210 grams, were enrolled in the study. Eight rats were included as a normal control group and did not receive any medication. 16 rats were administered cisplatin at a dosage of 2.5 mg/kg/day twice a week for 4 weeks (total dose 20 mg/kg). Then, they were randomly divided into two groups and treated with 1 ml/kg/day tap water or 8 mg/kg/day candesartan via oral gavage daily for 4 weeks. At the end of the treatment period, animals were sacrificed, and their kidneys were assessed histologically. In addition, plasma malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), creatinine, and GDF-15 levels were assessed. RESULTS: Treatment with candesartan resulted in a significant rise in serum GDF-15 levels and a significant reduction in levels of serum MDA, TNF-α, IL-6, and creatinine compared to the cisplatin and saline group. Candesartan treatment effectively protected the kidney injury, and histopathological examinations of the kidneys confirmed these results. CONCLUSIONS: This study demonstrates that candesartan alleviates cisplatin-induced renal toxicity by further increasing GDF-15, downregulating inflammatory markers, and reducing oxidative stress.

A pilot study for human tumor/DNA banking: returned more questions than answers.

A pilot study was performed for setting up the Dokuz Eylül University Breast Tumor DNA Bank (DEUBTB) to facilitate the sharing of tumor DNA/RNA samples and related data from cases collected by collaborators specializing in the breast cancer diseases between 2004 and 2006. The pilot study aimed to provide answers for certain questions on: (1) ethical concerns (informing the volunteer for donating specimen, anonymizing the sample information, procedure on sample request), (2) obtaining and processing samples (technical issues, flowchart), (3) storing samples and their products (storing forms and conditions), (4) clinical database (which clinical data to store), (5) management organization (quality and quantity of personnel, flowchart for management relations), (6) financial issues (establishment and maintenance costs). When the bank had 64 samples, even though it is quite ready to supply samples for a research project, it revealed many questions on details that may be answered in more than one way, pointing that all biobanks need to be controlled by a higher degree of management party which develops and offers quality standards for these establishments.

Effects of basic fibroblast factor (bFGF) on MMP-2, TIMP-2, and type-I collagen levels in human lung carcinoma fibroblasts.

Matrix metalloproteinases (MMP's) and tissue inhibitors of metalloproteinases (TIMP's) possess a preponderant role in the metabolism of the major extracellular matrix protein, collagen, and are thought to be important in the mechanism of tumor invasion. Lung cancer occupies the first position in mortality and the second position in incidence, among all cancers. In the present investigation, we studied the effect of basic fibroblast growth factor (bFGF) on collagen, matrix metalloproteinase-2 (MMP-2), and tissue metalloproteinase inhibitor-2 (TIMP-2) levels in normal and carcinoma lung tissue fibroblast cultures. MMP-2 was selected because of its high specificity in the degradation of type IV collagen, major component of the basal membrane. The effect of bFGF on MMP-2, TIMP-2, total collagen, and type I collagen levels of normal and carcinoma lung fibroblast cultures was investigated at 0, 10, and 100 ng/ml. Statistical analysis was carried out using the Mann-Whitney-U test and possible correlations were searched using the Spearman correlation analysis method. MMP-2, TIMP-2, total collagen, and type-1 collagen levels based on cell counts (10(3) cells) showed no statistically significant differences between the carcinoma and normal fibroblast cultures. However, positive correlations were found between MMP-2 and TIMP-2 in normal (P = 0.016) and carcinoma (P = 0.001) tissue fibroblast cultures. Positive correlations were also found between total collagen and TIMP-2 levels in normal and carcinoma tissue fibroblast cultures (P = 0.002 and P = 0.032). Total collagen and TIMP-2 levels also showed positive and strong correlations in all cultures except in 100 ng/ml bFGF concentrations. In addition, type I collagen and MMP-2 levels showed positive significant correlations only in normal and carcinoma control cultures, while type I collagen and TIMP-2 levels showed positive correlations in all cultures except carcinoma fibroblasts at 100 ng/ml bFGF. It may be concluded that bFGF does not affect MMP-2, TIMP-2, total collagen, and type-1 collagen levels in fibroblast cultures grown from human carcinoma and normal lung tissues. However, bFGF was noted, in vitro, to disturb the equilibrium which normally exists between the four parameters, both in normal and carcinoma tissue fibroblasts.

Human brain tumor phosphoglycerides.

This study is a qualitative and quantitative comparative analysis of the phosphoglycerides found in human brain tumors and normal brain tissue. Astrocytomas, glioblastomas, meningosarcomas and ependymomas were studied and, in all of these, the total phosphoglyceride levels were found to be significantly lower than in normal controls. Fractionation of the phosphoglycerides by thin-layer chromatography showed definite qualitative and/or quantitative differences in the phosphoglyceride fractions of tumor tissues in comparison with normal brain tissue.

Acid and alkaline phosphatase activities in homogenates and subcellular fractions of human brain tumors.

This study is a quantitative analysis of acid and alkaline phosphatase activity in human brain tumor homogenates and subcellular fractions, in parallel with normal brain tissue. Glioblastoma multiforme, meningioma, astrocytoma and normal tissue samples were separated by ultracentrifugation into five subcellular fractions: nuclei (N), mitochondria (M), microsomes (P), ribosomes (R) and supernatant (S). These two phosphatases showed significant increase in astrocytoma and meningioma tissue homogenates, compared with normal brain tissue. Alkaline phosphatase levels were determined to increase significantly in glioblastoma multiforme tissue homogenates as compared with normals, while those of acid phosphatase were observed to decrease. The results of this investigation also indicate that the subcellular distributions of acid and alkaline phosphatase show differences in the different tumor types. This observation is evidence against metabolic uniformity in tumoral tissue.

Hydrogen peroxide detoxication by catalase in subcellular fractions of human brain tumors and normal brain tissues.

Hydrogen peroxide detoxication by catalase was found to be significantly lower in human brain tumoral tissue as compared with normal brain tissue. As to the subcellular distribution of catalase activity, the tumoral tissue showed a decrease in the nuclear and mitochondrial fractions and an increase in the supernatants.

Cytosol and serum ferritin in breast carcinoma.

This study was undertaken to determine tissue and serum ferritin levels in different stages of breast carcinoma. Eighty-nine cases have been evaluated, the groups investigated being breast carcinoma, benign breast disease and healthy controls. Ferritin levels in both the sera and the tissue cytosols were measured by an enzyme immunoassay method, while total proteins were assayed by Lowry's procedure and the ferritin concentrations given in ng ferritin/mg cytosol protein. No significant difference has been determined for serum ferritin between any of the groups studied, while the tissue cytosol ferritins were found to be 91.6 +/- 50.9, 565.0 +/- 48.3, 142.7 +/- 93.3, 683.3 +/- 212.9 and 655.5 +/- 100.4 ng/mg cytosol protein for the benign, malign (global), malign (stage I), malign (stage II) and malign (stage III) groups, respectively. The differences between the malign groups and the benign group were found to be highly significant (P < 0.001) except for the stage I subgroup, which was fairly significant (P < 0.05). A sensitivity of 90% was evaluated for tissue cytosol ferritin in breast carcinoma, the 'intra-patient' sensitivity being 100%. In conclusion, we state that tissue ferritin is more valuable than serum ferritin as a tumour marker of diagnosis for breast carcinoma.

Evaluation of some antioxidant enzymes in lung carcinoma tissue.

This investigation was effected to determine the levels of the two antioxidant enzymes, superoxide dismutase (SOD) (EC 1.15.1.1) and catalase (CAT) (EC 1.11.1.6) in lung cancerous tissues and to compare with normal lung tissue in order to evaluate the antioxidant status in lung cancer. Fifteen lung carcinoma tissue samples and the normal counterparts from the same cases were homogenized and the cytosols obtained by ultracentrifugation (100,000 x g). SOD was assayed using a modification of the indirect nitroblue tetrazolium assay method, while CAT was measured by a spectrophotometric method. The data obtained are as follows: 1.42 +/- 0.24 U/mg protein (means +/- SEM) of SOD in lung cancer and 3.13 +/- 0.51 U/mg protein in normal lung tissue and 33.53 +/- 6.09 U/mg protein of CAT in lung cancer and 71.33 +/- 14.38 in normal lung tissue. The differences were found to be significant at the level of P < 0.01 for both enzymes. These low levels of the antioxidant enzymes in lung cancerous tissues can lead to elevated levels of reactive oxygen metabolites, resulting in damage to the key subcellular structures such as DNA, cell membranes, and other vital cellular components.

Determination of glutathione, glutathione reductase, glutathione peroxidase and glutathione S-transferase levels in human lung cancer tissues.

This study was performed to elucidate the lung glutathione-related defense potential in tumoral tissues. Reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase (GR), selenium-dependent (SeGPx) and total glutathione peroxidase (tGPx), and glutathione S-transferase (GST) activities in 38 tumoral lung tissues and 17 normal lung tissues were determined to obtain a comprehensive profile of the lung glutathione and glutathione-related enzymes in cancer. The enzyme levels in tumoral tissues (n = 38) were found to be significantly higher (P < 0.05) than those in normal tissues (n = 17). Reduced glutathione levels, and not oxidized glutathione levels, were found to be higher in normal tissues than those in tumoral tissues. We found no statistically significant difference between the adenocarcinoma and squamous cell carcinoma groups for any of the parameters studied.

Evaluation of lipid peroxidation, cathepsin L and acid phosphatase activities in experimental brain ischemia-reperfusion.

This investigation was conducted in rat brain tissues to elucidate the free radical induced cellular and subcellular membrane injuries in two different depth of global ischemia. Global moderate (penumbral) ischemia was performed on rat brains by bilateral vertebral arteries cauterization and temporary occlusion of the bilateral carotid arteries. Global severe ischemia was produced by a neck tourniquet in addition to four vessel occlusion. Somatosensory evoked potentials (SSEPs) were used as a feed back parameter to monitor electrophysiologically the ischemia. At the end of ischemic insult (0 min reperfusion) or various reperfusion periods (20, 60 and 240 min), all rats were decapitated and brains were frozen in liquid nitrogen. The brain tissues were prepared for the determination of cathepsin L (CL) and acid phosphatase (AP) activities in the supernatant (cytosolic) fraction (SF) and the fraction enriched with lysosomes (FEL). Further the level of thiobarbituric acid reactive substances (TBARS) of lipid peroxidation was assessed by the spectrophotometric methods. Severe ischemia-reperfusion was accompanied by a significant increase in TBARS levels and the SF/FEL ratio for CL and AP activities compared to the sham operated group and the concurrent reperfusion groups of moderate ischemia (p<0.05). There were no significant differences between the sham operated and moderate ischemia-reperfusion groups for the same parameters. Our data clearly demonstrate that; in rat brain although severe ischemia-reperfusion causes lipid peroxidation in cellular membranes and redistribution of lysosomal enzymes from lysosomes to cytoplasm due to lysosomal membrane injury, there are no changes in lysosomal membrane stability in moderate ischemia-reperfusion.

The inversely proportional relation between nitric oxide and lipid peroxidation in atherosclerotic plaque formation in human.

It is known that in the presence of oxygen radicals, anti-atherogenic nitric oxide is converted into pro-atherogenic products, which increase lipid peroxidation. In this study, plaque-free atherosclerotic tissues (n=26), atherosclerotic plaques (n=26) and fetal tissues (n=2; as control) were evaluated. High nitrite, but low malondialdehyde, levels in non-atherosclerotic tissues may show the protective role of nitric oxide from atherosclerosis. In plaque-developed tissues nitrite levels were three times, and lipid peroxidation levels were 10 times, higher than non-plaque developed tissues. In the atherosclerotic plaque forming process, the role of nitric oxide can be discovered according to the lipid peroxidation of tissues. In conclusion, the results of this study show an inversely proportional relation between pro- and anti-atherogenic effects of nitric oxide in the pathogenesis of atherosclerotic vascular diseases.

Erythrocyte membrane and cytoskeletal protein glycation and oxidation in short-term diabetic rabbits.

The objective of this study was to elucidate the glycation and oxidation processes in plasma and erythrocyte membrane proteins as well as the major erythrocyte cytoskeletal protein, spectrin, using a short-term experimental rabbit diabetes model. Diabetes was induced with a single-dose alloxan injection. Spectrin was purified from erythrocyte ghosts with selective solubilization followed by gel filtration chromatography techniques, and tested for purity using sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. Glycation in plasma proteins was measured as fructosamine using the nitroblue tetrazolium method, and in erythrocyte membrane and purified spectrin, as ketoamine equivalents, by the hydrazine/phenylhydrazine method. Protein oxidation in plasma, erythrocyte membrane proteins, and purified spectrin was evaluated in terms of sulfhydryl oxidation, based on cis-dichlorodiammine platinum (II) binding. Carbonyl formation was also measured in plasma and membrane proteins. Sulfhydryl oxidation, carbonyl groups and glycated protein levels showed statistically significant differences between the diabetic and control groups for both the plasma and the erythrocyte membrane proteins. The cis-dichlorodiammine platinum (II) binding was significantly different in diabetic rabbit erythrocyte spectrin, while glycation was not significantly different for this protein. Our data clearly demonstrate that both protein glycation and oxidation are biochemical alterations occurring in diabetes, even of short duration.

Lipoprotein profile in long term theophylline administration in children with asthma.

Atherosclerosis in childhood has a slowly progressive course and its clinical features usually become prominent in middle ages. Hypercholesterolemia is one of the major risk factors for the development of atherosclerosis. A clear correlation exists between hypercholesterolemia in childhood and atherosclerotic lesions extending into adulthood.In this study, we evaluated the effect of slow release theophylline (SRT) treatment on plasma lipid profile and assessed the risk for atherosclerotic coronary heart disease in children with bronchial asthma. Group 1 consisted of 15 children with a mean age of 10.8 3.19 years who received SRT for bronchial asthma for a mean period of 9.13 2.17 months. Group 2 was composed of 15 children with a mean age of 11.40 3.78 years and followed up for bronchial asthma, who received no SRT treatment. Group 3 comprised 15 children with a mean age of 9.00 3.76 years and no history of asthma or wheezing. In all patients lipid profiles were assessed by measuring levels of plasma triglyceride, total cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) apolipoprotein A (Apo-A) and apolipoprotein B (Apo-B). In group 1, the mean total cholesterol level was 175.53 24.36 mg/dl, LDL-C level was 91.00 24.07 mg/dl and Apo-B level was 87.27 12.74 mg/dl after SRT treatment. In group 1, group 2 (control group with asthma) and group 3 (the non-asthmatic control group), the mean plasma lipid level after SRT treatment was significantly higher than that before SRT treatment. In conclusion, long-term SRT treatment in children with bronchial asthma may alter lipid profile and may increase the risk for developing atherosclerotic coronary heart disease.

Biochemical analysis of total collagen content and collagen types I, III, IV, V and VI in gingiva of various periodontitis categories.

Collagen is one of the chief components of the extracellular matrix of gingival connective tissue, where five different types have been identified to date. The molecular mechanism of collagen loss in periodontitis still needs to be explored. In the present study total collagen content was investigated in gingival connective tissue of adult periodontitis (AP) as well as early onset periodontitis patients (EOP) and clinically healthy subjects. Furthermore, collagen type I, III, IV, V andVI content was evaluated in gingival biopsies obtained from periodontitis patients. There was a statistically significant difference between AP (25.1 +/- 8.1 microg/mg) and EOP (15.6 +/- 4.0microg/mg) groups with regard to the total collagen content (P < 0.05). In the clinically healthy control group the total collagen content was 20.7 +/- 4.6microg/mg. Moreover, the distribution of collagen types exhibited variations in pooled homogenates of each periodontitis group. The total collagen loss seemed to be greater in the EOP patients than in the AP patients. When the ratio of fibril forming collagens to nonfibrillar collagens was evaluated, it seems to be decreased in AP patients in comparison to EOP patients. The findings of the present study suggest that different collagen types present in various periodontitis categories may be related with diverse pathogenic mechanisms acting in these diseases.

The effects of alpha-lipoic acid on MMP-2 and MMP-9 activities in a rat renal ischemia and re-perfusion model.

Matrix metalloproteinases (MMPs) are enzymes that are responsible for degradation of extracellular matrix (ECM); they are involved in the pathogenesis of ischemia-re-perfusion (I-R) injury. We investigated the possible preventive effect of alpha-lipoic acid (LA) in a renal I-R injury model in rats by assessing its reducing effect on the expression and activation of MMP-2 and MMP-9 induced by I-R. Rats were assigned to four groups: control, sham-operated, I-R (saline, i.p.) and I-R+ LA (100 mg/kg, i.p.). After a right nephrectomy, I-R was induced by clamping the left renal pedicle for 1 h, followed by 6 h re-perfusion. In the sham group, a right nephrectomy was performed and left renal pedicles were dissected without clamping and the entire left kidney was excised after 6 h. LA pretreatment was started 30 min prior to induction of ischemia. Injury to tubules was evaluated using light and electron microscopy. The expressions of MMP-2 and MMP-9 were determined by immunohistochemistry and their activities were analyzed by gelatin zymography. Serum creatinine was measured using a quantitative kit based on the Jaffe colorimetric technique. Malondialdehyde (MDA) and glutathione (GSH) were analyzed using high performance liquid chromatography. Tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-1 were assessed using enzyme-linked immunosorbent assay (ELISA). I-R caused tubular dilatation and brush border loss. LA decreased both renal dysfunction and abnormal levels of MDA and GSH during I-R. Moreover, LA decreased significantly both MMP-2 and MMP-9 expressions and activations during I-R. TIMP-1 and TIMP-2 levels were increased significantly by LA administration. LA modulated increased MMP-2 and MMP-9 activities and decreased TIMP-1 and TIMP-2 levels during renal I-R.

Ferritin: a tumor marker expressed by renal cell carcinoma.

Renal cell carcinoma (RCC) has been shown to secrete several hormones and biologically active substances that influence the host metabolism or induce paraneoplastic syndromes. Observation of anemia in 20% of patients with RCC and the spontaneous recovery of anemia following nephrectomy drew attention to the body iron metabolism. Ferritin was previously proposed as a tumor marker for RCC. In order to determine whether RCC cells actually produce ferritin, we studied ferritin levels in serum from peripheral and renal veins as well as from the tumor tissue and the healthy parenchyma from radical nephrectomy specimens of 22 patients with RCC. Ferritin levels both in sera and cytosols were measured by an enzyme immunoassay method. The mean serum ferritin level from the renal vein was 419.9 +/- 72.4 ng/ml, and it was 157.3 +/- 18.3 ng/ml from the peripheral vein (p < 0.05). Renal vein ferritin correlated with stage and had a significant impact on prognosis (p < 0.05). The mean cytosolic ferritin level of the cancer tissue was 705.6 +/- 56.9 ng/mg cytosol protein, whereas in the normal parenchyma it was 95.9 +/- 10.1 ng/mg cytosol protein. This was also highly significant (p = 1.15 x 10(-13)), suggesting that RCC cells probably express ferritin. As currently there exists no reliable tumor marker for RCC, the value of ferritin as a marker should be investigated further before drawing any clinical conclusions.

Arthroscopically assisted tibial plateau fracture management: a modified method.

We present a new technique for arthroscopically assisted treatment of tibial plateau fracture. This is a modification of previously described reduction and grafting techniques. We performed a core graft under the depressed fragment and used it for reduction. With the aid of anterior cruciate ligament surgery techniques, we aimed to simplify the graft harvesting and reduction for better healing.

Anterior sacroiliac fusion: a new video-assisted endoscopic technique.

We present a case of sacroiliac fusion performed for an intraarticular osteochondroma of the sacroiliac joint, which was the cause of severe pain and disability. Excision of the lesion and sacroiliac fusion were successfully performed by utilizing minimally invasive surgical techniques. Although the application of this technique requires a dedicated and highly experienced team, the encouraging result of our first case, with minimal morbidity and disability due to the operation, induces us to recommend this technique in sacroiliac fusion, especially when fusion is combined with additional procedures such as drainage, biopsy, or excision.

Alterations in superoxide dismutase, glutathione peroxidase and catalase activities in experimental cerebral ischemia-reperfusion.

Free radicals are thought to be the most important cause of the reperfusion injury subsequent to ischemia. The antioxidant status of the tissue affected by ischemia-reperfusion is of great importance for the primary endogenous defense against the free radical induced injury. This investigation was performed to evaluate the antioxidant enzyme capacity of the brain tissue in the ischemia-reperfusion period using an experimental global moderate (penumbral) ischemia model on rat brains. Experiments were performed on 45 male Sprague Dawley rats. Ischemia was induced by bilateral vertebral arteries cauterization and temporary bilateral carotid arteries occlusion and sustained for 10 minutes. At the end of ischemia (0 min reperfusion) and various reperfusion periods (20 min, 60 min, 240 min), rats were decapitated and brains were frozen in liquid nitrogen. Changes in the intracellular antioxidant enzyme (superoxide dismutase, glutathione peroxidase and catalase) activities were assessed in the rat brain tissues, by spectrophotometric methods. In all moderate ischemia-reperfusion groups, superoxide dismutase activities were found to have decreased significantly compared to the sham operated controls (P < 0.05). During ischemia superoxide dismutase activity was lowered to 31% of that of the control group. The decreases were more significant in reperfusion groups, particularly in 60 min reperfusion (40%). Relatively smaller but still significant diminution was observed in glutathione peroxidase activities (P < 0.05). The ratio of diminution was striking in 20 min and 60 min reperfusion groups with 26% of the sham operated rats. Conversely, moderate ischemia-reperfusion caused significant increase in catalase activities (P < 0.05). The increment was 63% of the preischemic level with 10 min of moderate ischemia. In conclusion, activities of the major antioxidant enzymes were changed significantly in moderate brain ischemia-reperfusion. These results suggest that the disturbance in oxidant-antioxidant balance might play a part in rendering the tissue more vulnerable to free radical induced injuries.