Pharmacological treatment of oro-facial pain - health technology assessment including a systematic review with network meta-analysis.

This health technology assessment evaluated the efficacy of pharmacological treatment in patients with oro-facial pain. Randomised controlled trials were included if they reported pharmacological treatment in patients ≥18 years with chronic (≥3 months) oro-facial pain. Patients were divided into subgroups: TMD-muscle [temporomandibular disorders (TMD) mainly associated with myalgia]; TMD-joint (TMD mainly associated with temporomandibular joint pain); and burning mouth syndrome (BMS). The primary outcome was pain intensity reduction after pharmacological treatment. The scientific quality of the evidence was rated according to GRADE. An electronic search in PubMed, Cochrane Library, and EMBASE from database inception to 1 March 2017 combined with a handsearch identified 1552 articles. After screening of abstracts, 178 articles were reviewed in full text and 57 studies met the inclusion criteria. After risk of bias assessment, 41 articles remained: 15 studies on 790 patients classified as TMD-joint, nine on 375 patients classified as TMD-muscle and 17 on 868 patients with BMS. Of these, eight studies on TMD-muscle, and five on BMS were included in separate network meta-analysis. The narrative synthesis suggests that NSAIDs as well as corticosteroid and hyaluronate injections are effective treatments for TMD-joint pain. The network meta-analysis showed that clonazepam and capsaicin reduced pain intensity in BMS, and the muscle relaxant cyclobenzaprine, for the TMD-muscle group. In conclusion, based on a limited number of studies, evidence provided with network meta-analysis showed that clonazepam and capsaicin are effective in treatment of BMS and that the muscle relaxant cyclobenzaprine has a positive treatment effect for TMD-muscle pain.

Influence of age on the metabolism of plasma low density lipoproteins in healthy males.

The plasma concentration of the atherogenic low density lipoproteins (LDL) increases with age. To clarify the mechanism of this change, we studied the kinetics of autologous 125I-LDL apolipoprotein B (apo B) in 41 normolipidemic, nonobese healthy males. For comparison, they were divided into three age groups: young, 21-39 yr (n = 18), middle-aged, 40-59 yr (n = 11), and old, 60-80 yr (n = 12). The levels of plasma LDL cholesterol and LDL apo B increased from respectively 3.4 +/- 0.1 (SEM) mmol/liter and 86 +/- 2 mg/dl in the young to 4.1 +/- 0.1 mmol/liter and 95 +/- 3 mg/dl in the old (P less than 0.01), and this increase was linked to a progressively decreased (r = -0.38, P less than 0.02) fractional catabolic rate of LDL apo B (0.348 +/- 0.010 pools per day in the young vs. 0.296 +/- 0.009 pools per day in the old, P less than 0.01). The production rate of LDL apo B did not differ significantly between the groups. The reduced fractional catabolic rate of LDL apo B in the old was not associated with a decrease in binding affinity of the LDL particle to its receptor, as judged from its ability to compete for 125I-LDL fibroblast binding. When hepatic LDL receptor expression was stimulated by cholestyramine treatment in six old males, their LDL apo B fractional catabolic rate increased to the levels observed in the young subjects. We conclude that the increase in LDL which normally occurs with age is explained by a reduced capacity for its removal, and hypothesize that this is mediated via a reduced hepatic LDL receptor expression.

Low-density lipoprotein as a carrier of antitumoral drugs: in vivo fate of drug-human low-density lipoprotein complexes in mice.

Previous studies have demonstrated that human leukemic cells and certain cancer cells in culture have a higher uptake of plasma low-density lipoprotein (LDL) than the corresponding normal cells. Therefore LDL has been proposed as a drug carrier for anticancer agents. In the present investigation, we have developed a method to incorporate a lipophilic derivative of doxorubicin, N-trifluoroacetyladriamycin-14-valerate, into LDL. The method involves lyophilization of LDL in the presence of sucrose as protective agent and gives an N-trifluoroacetyladriamycin-14-valerate-LDL complex containing about 100 drug molecules per LDL particle. The in vivo fate of the complex in mice as judged from the disappearance from plasma and accumulation in organs was similar to that of native LDL. When cultured human fibroblasts were incubated with N-trifluoroacetyladriamycin-14-valerate-LDL, cellular drug accumulation was dependent on the LDL receptor activity of the cells. The covalent linkage of two anthracycline derivatives to lysine residues of LDL yielded conjugates with drug/LDL molar ratios ranging up to 80. With increasing substitution, there was a progressive decline in the affinity of the conjugate for the LDL receptor in vitro. The in vivo fate of such conjugates was quite similar to that of native LDL. We conclude that it is possible to associate cytotoxic agents with LDL without interfering with its in vivo behavior.

Elevated uptake of low density lipoproteins by human lung cancer tissue in vivo.

In order to explore new treatment modalities for cancer, it is important to identify qualitative or quantitative differences in metabolic processes between normal and malignant cells. Previous in vitro and in vivo studies have shown that acute myelogenous leukemia cells have elevated receptor-mediated uptake of low density lipoproteins (LDL), compared to normal WBC. High receptor-mediated uptake of LDL by certain cancer cells in tissue culture and experimental tumors in animals in vivo has also been demonstrated. The present study was undertaken to compare the in vivo assimilation of LDL by human lung cancer tissue with that by surrounding lung tissue. Ten patients with newly diagnosed lung tumors, scheduled for surgery, received an i.v. injection of [14C]sucrose-labeled LDL. Following cellular uptake and degradation of the LDL particle, the radiolabeled sucrose moiety remains trapped in the lysosomal compartment, making this labeling technique useful for in vivo studies of tissue uptake of LDL. Radioactivity was determined in plasma and in tissue biopsies obtained at surgery 1-3 days after injection. The uptake of radioactivity in lung cancer tissue was elevated (1.5-3.0-fold), compared to surrounding tissue, in 7 of 9 patients with primary lung cancer. The most rapid preoperative disappearance of radioactivity from plasma was found in 2 patients with large tumors exhibiting high LDL uptake, relative to normal lung tissue. These findings support the hypothesis that the selectivity of cytotoxic agents can be enhanced also in nonhematological malignancies by administering the drugs incorporated in LDL particles.

Selective uptake of a toxic lipophilic anthracycline derivative by the low-density lipoprotein receptor pathway in cultured fibroblasts.

N-(N-Retinoyl)-L-leucyldoxorubicin 14-linoleate (r11-DOX), a new lipophilic derivative of doxorubicin, was synthesized and incorporated into low-density lipoprotein (LDL). The drug-LDL complex contained 100-200 drug molecules/LDL particle. When cultured normal human fibroblasts were incubated with 125I-LDL-incorporated drug, there was a perfect correlation between the cellular uptake plus degradation of 125I-LDL and the cellular drug accumulation. The presence of excess native LDL inhibited the cellular uptake and degradation of 125I-LDL and the drug accumulation to the same extent. In contrast, methylated LDL, which does not bind to the LDL receptor, did not alter the cellular uptake and degradation of 125I-LDL nor did it alter the drug accumulation. When LDL receptor negative fibroblasts from a patient with the homozygous form of familial hypercholesterolemia were incubated with the drug-125I-LDL complex, cellular drug accumulation was very low. The drug-LDL complex inhibited the growth of cultured normal human fibroblasts. The drug incorporated into methylated LDL was much less toxic. These findings suggest that r11-DOX incorporated into LDL is delivered to cells selectively by the LDL receptor pathway. This might be of value in the treatment of leukemia, since we have previously found that leukemic cells exhibit higher LDL receptor activity than white blood cells and bone marrow cells from healthy subjects.

Cholesterol catabolism in patients with acute myelogenous leukemia and hypocholesterolemia: suppressed levels of a circulating marker for bile acid synthesis.

Hypocholesterolemia is a frequent finding in patients with acute myelogenous leukemia (AML) and in other types of malignancies. Since bile acids are major excretion products of cholesterol, the hepatic degradation of cholesterol to bile acids was investigated in AML patients by analyzing a circulating marker for bile acid synthesis. In addition, plasma levels of a marker for cholesterol synthesis were determined. The plasma levels of 7alpha-hydroxy-4-cholesten-3-one, reflecting bile acid production, were markedly lower in patients with AML than in healthy controls. The median levels were 3.3 and 18.5ng/ml (P<0.0001) in the AML patients (n=29) and the healthy subjects (n=16), respectively. The plasma levels of 7-dehydrocholesterol, reflecting hepatic cholesterol synthesis, were similar for the AML patients and the controls. The results show that the conversion of cholesterol to bile acids was suppressed in AML patients, a phenomenon that may result in a decreased intestinal absorption of cholesterol and subsequent hypocholesterolemia.

Expression of the low-density lipoprotein receptor, HMG-CoA reductase, and multidrug resistance (Mdr1) genes in colorectal carcinomas.

Some malignant cells have elevated uptake of plasma low-density lipoprotein (LDL). We determined the expressions in colorectal cancers of the LDL receptor gene, of the gene for the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and of the multidrug resistance gene (mdr1) by quantitative RNA-RNA solution hybridisation. LDL receptor RNA levels in tumor tissue exceeded those in normal mucosa in 20 of 23 patients (2-11-fold higher in 17 of 23 patients), with a mean +/- SD of 7.8 +/- 5.8 copies/cell in tumor tissue vs 3.5 +/- 2.5 in normal mucosa (P = 0.002). The HMG-CoA reductase gene was similarly expressed in tumor and normal tissue, with means and SD of 2.0 +/- 1.3 copies/cell versus 2.2 +/- 1.9 (pi = 21). Mdr1 RNA was undetectable ( < 0.15 copies/cell) in 5 of 20 tumors, with a mean +/- SD of 1.0 +/- 1.1 copies/cell vs 1.6 +/- 1.7 in normal mucosa. Expression of all three genes was, in most cases, higher in normal liver than in liver metastasis of colorectal carcinomas or normal colon mucosa. The results may form the basis for using LDL as a drug carrier for treatment of colorectal carcinomas, and may indicate that drug resistance in these tumors is not due to overexpression of the mdr1 gene.

Effect of verapamil on daunorubicin accumulation in human leukemic cells with different levels of MDR1 gene expression.

The purpose of the present study was to evaluate the distribution of the multiple drug resistance phenotype in leukemic cells with different levels of mdr1 gene expression. Peripheral leukemic cells from 8 patients (5 with detectable and 3 with undetectable mdr1 RNA levels) were incubated with daunorubicin (1 microM) in the absence and presence of verapamil (6.6 microM). Daunorubicin accumulation in individual cells was analysed by flow cytometry. Verapamil increased (16-45%) the mean daunorubicin accumulation in 7 of the 8 cell samples. In cells from 4 of these 7 patients, the fluorescence histograms showed a general increase of daunorubicin accumulation. The increase was limited to a small subpopulation of cells in one patient. In the remaining two patients an increase of daunorubicin accumulation was seen in the majority of cells, while cells with the highest accumulation were unaffected. In conclusion, verapamil can increase daunorubicin accumulation in leukemic cells with and without detectable mdr1 gene expression and the increase seems to affect the majority of the cell population.

Multidrug resistance (Mdr1) gene expression in peripheral blasts from patients with acute leukemia only rarely increases during disease progression after combination chemotherapy.

Multidrug resistance gene (mdr1) RNA levels were determined in 55, and P-glycoprotein expression in 37 samples of peripheral leukemic cells from 17 patients with acute myeloblastic leukemia (AML) and 7 patients with acute lymphocytic leukemia (ALL). Between sample collections, patients were treated with various chemotherapy regimens. Mdr1 RNA levels were quantified by a RNA-RNA solution hybridization assay. P-glycoprotein was determined by Western blot analysis. Samples from 14 patients (9 AML, 5 ALL) had undetectable mdr1 RNA levels at initial analysis. Only two of these had detectable levels after chemotherapy. Ten patients (8 AML, 2 ALL) had detectable mdr1 RNA levels at initial analysis (median 1.0 transcript per cell, range 0.2-1.4). Increase of mdr1 RNA levels after chemotherapy were observed in cells from 3 patients, one patient had a lower level after chemotherapy and the 6 remaining patients had essentially unchanged mdr1 RNA levels in their leukemic cells. Samples from 13 patients were sequentially analysed for P-glycoprotein expression. In one patient, no P-glycoprotein was detectable at initial analysis but was weakly positive after chemotherapy. In the remaining 12 patients, P-glycoprotein levels stayed stable during disease progression. In conclusion, combination chemotherapy seems only rarely to be associated with an increase of mdr1 gene expression in residual leukemic cells. The addition of resistance modifiers to chemotherapy in order to overcome P-glycoprotein mediated resistance might therefore be more effective in chemotherapy naive patients since it is possible that during later disease progression additional mechanisms of resistance may be more operative.

Low density lipoprotein as a carrier of cytostatics in cancer chemotherapy: study of stability of drug-carrier complexes in blood.

Several solid tumour and leukemia cell types have a higher low density lipoprotein (LDL) uptake than the corresponding normal cells. We are investigating the possibilities to use LDL as a drug carrier to increase the selectivity of antineoplastic drugs in cancer chemotherapy. We have developed a method to incorporate lipophilic cytotoxic agents without interfering with the in vitro and in vivo properties of LDL. In this study, we examined the stability of some drug-LDL complexes in blood and plasma as this is an important prerequisite to achieve a selective therapy. The in vitro dialysis of N-trifluoroacetyl-adriamycin-14-valerat-LDL (AD-32-LDL) against plasma revealed a slow dissociation of the complex. The same method showed a fast and total leakage of paclitaxel from paclitaxel-LDL into the plasma chamber. The dissociation of paclitaxel was confirmed by an autoradiographic study of the distribution of paclitaxel-LDL in tumour-bearing mice. In patients with leukemia the rapid plasma dissociation of AD-32 from LDL illustrated a much higher in vivo instability of this complex. With this method, cholesteryl-linoleate only could be incorporated into LDL in a stable manner as shown by dialysis and autoradiography results. The incorporation of cytotoxic drug derivatives, containing lipophilic anchors, is now under study in order to obtain LDL complexes with better plasma stability.

Plasma stability and cytotoxicity of lipophilic daunorubicin derivatives incorporated into low density lipoproteins.

The selective targeting of antineoplastic drugs to tumours by incorporation in low density lipoproteins (LDL) is an attractive possibility if the drug-LDL complex remains stable in the circulation and is taken up by the tumour. In previous studies we have shown that vincristine- and N-trifluoroacetyladriamycin-14-valerate-LDL complexes were unstable in vivo. We synthesized five N-substituted lipophilic derivatives of daunorubicin and studied their incorporation into LDL. Three out of five daunorubicin derivatives incorporated successfully into LDL. In vitro these complexes were more cytotoxic towards LDL receptor positive Chinese hamster ovary cells than LDL receptor negative cells. Non-specific cytotoxicity was explained by slow dissociation of the drug-LDL complex in plasma. Our results underline the importance of careful studies of plasma stability when investigating lipoproteins and other carriers in drug targeting.

Multidrug resistance gene (mdr1) RNA levels in relation to P-glycoprotein content of leukemic cells from patients with acute leukemia.

The clinical relevance of multidrug resistance gene (mdr1) expression in tumor cells remains largely unclear. Conflicting results regarding mdr1 gene expression and clinical outcome have been obtained. Little is known about regulation of mdr1 gene expression, and the conflicting results might be explained by the fact that mdr1 RNA levels do not reflect expression at the protein level. The aim of the present study was to investigate the relationship between mdr1 RNA levels and P-glycoprotein (Pgp) content of leukemic cells from patients with acute myelogenous or lymphocytic leukemia. Mdr1 RNA levels were determined by a quantitative RNA-RNA solution hybridization method, and Pgp by Western blot technique with enhanced chemiluminescence for immunodetection. Pgp was detected in 14/14 leukemic cell samples while mdr1 RNA was detectable (> 0.15 copies/cell) in cells from only six out of the 14 patients. Mdr1 RNA levels did not correlate with the Pgp content of leukemic cells (r = 0.284, p = 0.306). Relapsed leukemias had significantly (p = 0.016) higher levels of Pgp than de novo untreated leukemias (the mean and SD optical density units were 0.56 +/- 0.18 and 0.25 +/- 0.17 respectively) while no difference was found in RNA levels. The findings support post-transcriptional level regulation of mdr1 gene expression and stress the importance of accurate determinations of the Pgp content of tumor cells in studies of the relationship between mdr1 gene expression and clinical outcome.

Growth modulation of low density lipoprotein receptor sterol sensitivity in cultured human fibroblasts.

Sterols regulate low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene expressions by end product repression. Studies on cultured cells have shown that growing cells have a higher LDL uptake than quiescent cells and that incubation of cells with growth factors or mitogenic compounds leads to sterol-resistant upregulation of LDL receptor gene expression. The recent finding that elevated LDL receptor activity in acute myelogenous leukemia cells was characterized by a decreased sensitivity to downregulation by sterols raises the possibility that the mechanism behind this is related to the cellular growth rate. By using cultured human fibroblasts as a model system we therefore studied whether growth modulation of sterol sensitivity takes place in normal actively growing cells. Judging from the ability of sterols (25-hydroxycholesterol + cholesterol) to inhibit 125I-LDL degradation, we found that the sensitivity to sterols varied markedly between cells of different densities. The lowest sensitivity to sterols and highest 125I-LDL degradation rate were found in subconfluent cells, whereas sparse and confluent cells were the most sensitive ones. In contrast to the LDL receptor, HMG-CoA reductase sterol sensitivity did not appear to be growth regulated. We conclude that growth-dependent modulation of sterol sensitivity and LDL receptor activity takes place in normal human fibroblasts. Modulation of sterol sensitivity may be an important mechanism to ensure an adequate cholesterol supply in growing cells.

Simvastatin impairs mitogen-induced proliferation of malignant B-lymphocytes from humans--in vitro and in vivo studies.

Chronic lymphocytic leukemia (CLL) cells express lower low density lipoprotein (LDL) receptor activity and higher 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity than normal mononuclear blood cells indicating that CLL cells may depend on cholesterol synthesis for their proliferation. We studied the effects of competitive inhibitors of HMG-CoA reductase on malignant lymphocyte proliferation in vitro and in vivo. Tumor B-cells from 13 patients with CLL, hairy cell leukemia, or immunoblastic B-cell lymphoma were cultured for 4 d in the presence of B-cell mitogens and cholesterol synthesis inhibitors. Simvastatin and lovastatin suppressed, in a concentration-dependent manner, the mitogen-induced cellular thymidin uptake in medium with 10% human AB-serum or lipoprotein-deficient serum. Pravastatin was active only in medium with lipoprotein-deficient serum. Ten previously untreated patients with CLL received simvastatin orally, 40 mg daily for 12 wk. Mean reductions in total plasma and LDL cholesterol were 30% (range 9-46%) and 37% (range 16-63%), respectively. Cells from four patients showed moderate to minor increases in the degradation rate of 1251-LDL suggesting that the need for exogenous cholesterol had increased, three patients showed an increase in HMG-CoA reductase activity, and the cells from one patient showed both. There was no significant change in the clinical disease status during medication. However, four of the ten patients developed a therapy-demanding progressive disease during the subsequent year. Further clinical studies with cholesterol synthesis inhibitors in leukemia are warranted.

Uptake of low-density lipoprotein by malignant cells--possible therapeutic applications.

Cells acquire cholesterol via de novo synthesis and receptor-mediated uptake of low-density lipoprotein (LDL), the major cholesterol-carrying lipoprotein in blood. Human leukemic cells and certain tumor tissues display elevated receptor-mediated uptake of LDL as compared with the corresponding normal cells or tissues. LDL has therefore been proposed as a potential carrier for chemotherapeutic agents. Various methods have been employed to incorporate antineoplastic lipophilic drugs into LDL, and the resultant drug-LDL complexes have been shown to be cytotoxic toward tumor cells in vitro. Initial experiments with tumor-bearing animals suggest that LDL may be a promising carrier for drugs in the treatment of malignant diseases.