Treatment with laquinimod reduces development of active MRI lesions in relapsing MS.

BACKGROUND: Laquinimod is a novel immunomodulatory substance developed as an orally available disease modifying treatment in multiple sclerosis (MS). The purpose of this study was to evaluate safety, tolerability, and efficacy on MRI lesions of two different doses of laquinimod compared with placebo in patients with relapsing MS. METHODS: In this multicenter, double-blind, randomized trial, patients with relapsing MS received 0.1 mg or 0.3 mg laquinimod or placebo as three daily tablets for 24 weeks. Gadolinium-enhanced brain MRI scans were performed at screening, every eighth week during treatment, and 8 weeks after end of treatment. The primary efficacy variable was the cumulative number of active lesions over 24 weeks. Safety measures included adverse events, physical examination, and laboratory variables. RESULTS: Of 256 screened patients, 209 were randomized (67 to 74 patients per group) in 20 centers. There was a significant difference between laquinimod 0.3 mg and placebo for the primary outcome measure (mean cumulative number of active lesions reduced by 44%). In the subgroup of patients with at least one active lesion at baseline the reduction was slightly more pronounced (52%). No differences with respect to clinical variables (relapses, disability) were found. The safety profile was favorable; there were no clinical signs of undesired inflammatory manifestations. CONCLUSION: Oral laquinimod in a dosage of 0.3 mg daily was well tolerated and effective in suppressing development of active lesions in relapsing multiple sclerosis.

Binding of epidermal growth factor (EGF) to a cultured human glioma cell line.

We have studied binding of 125I-EGF to the human malignant glioma cell line U-343 MG aCl2:6, which is planned to be used as a model system in studies of toxic effects of EGF conjugates. Special care has been taken to fulfil the requirements for a correct Scatchard analysis of binding parameters. Binding as a function of time, temperature and pH was investigated as well as dissociation and internalization of bound EGF. The stability of EGF during incubation was also determined. After binding to the receptor, EGF is rapidly internalized and degraded at physiological temperature. We found that binding experiments should be performed at 4 degrees C, since at this temperature practically no internalization took place, whereas dissociation occurred. From displacement experiments using increasing concentrations of unlabelled EGF competing with 125I-EGF for binding, binding parameters were calculated using a computerized, nonlinear, least-squares regression analysis of binding data. We found that EGF bound to a class of high affinity receptors with an apparent dissociation constant KD of about 4 x 10(-10) M. The mean number of receptors was 25,000 per cell. In experiments where receptors were saturated with 125I-EGF an additional class of low affinity receptors was detected. This had an apparent KD of 1 x 10(-8) M with a mean receptor number per cell of 780,000. We also noticed enhanced dilution-induced dissociation of bound 125I-EGF in the presence of excess unlabelled EGF, suggesting negative cooperativity.

An in vitro bioassay for quantitation of human interferons by measurements of antiproliferative activity on a continuous human lymphoma cell line.

Interferons have, in addition to their antiviral effects, been shown to possess several non-antiviral activities. In this study, an in vitro bioassay for interferon alpha (IFN-alpha) preparations based on their antiproliferative effect in cultured Daudi cells has been developed. Briefly, about 10(5) cells per ml treated with different concentrations of IFN were incubated under standard culture conditions for 3 days. Two different end points, i.e. incorporation of [3H]thymidine and final cell density, were used and responses were evaluated according to established pharmacopoeial principles for quantification of biomolecules. Both methods gave similar results. However, measurement of final cell density yielded the most precise results. The proposed assay, with an effective assay range of 1-10 IU/ml (approximately 0.2-2 x 10(-12)M, had a high sensitivity and precision as well as a good reproducibility. Compared with antiviral assays, it is less resource demanding. In conclusion, the in vitro bioassay described is well suited for potency determinations of IFN-alpha and probably also IFN-beta preparations.

In-vitro bioassays for cholecystokinin.

Two in-vitro methods for the bioassay of cholecystokinin (CCK) are described. They should be useful for determinations of potencies of pharmaceutical formulations of CCK. Both are based on the hormone's ability to stimulate exocrine pancreas. The stimulatory effect on 45Ca efflux from preloaded suspended acinar cells or on release of amylase from pancreatic acini from the guinea-pig has been recorded. The assay based on stimulation of 45Ca outflux is more specific and precise than the method using measurements of amylase secretion. Therefore, the calcium assay was further validated against the conventional guinea-pig gall-bladder contraction assay. Both methods gave similar potency readings but the results from the in-vitro assay were more precise.

Extracellular matrices in multicellular spheroids of human glioma origin: increased incorporation of proteoglycans and fibronectin as compared to monolayer cultures.

Tumor spheroids were cultured from five human glioma cell lines which differed considerably in their relative amount and composition of glycosaminoglycans (GAG), fibronectin and other extracellular matrix (ECM) components when grown as monolayer cultures. These differences were also evident when the cells were grown as spheroids. Under the 3-dimensional geometry of the spheroid system, there was, however, generally a more extensive ECM. Especially noteworthy was the presence of a small proteoglycan, probably a dermatan sulphate proteoglycan, in the ECM of the spheroids, but not in the monolayers. Noteworthy was also the appearance of fibronectin in spheroids which did not show any staining for fibronectin when grown as monolayer. The two spheroid types (U-87MG, U-105MG) with the most extensive matrix, and with the lowest proportion of hyaluronic acid (HA), had a low proliferation rate, whereas the three other spheroid types (U-118MG, U-138MG, U-251MG) with a less extensive ECM, and a relatively high production of HA had a much higher proliferation rate. These data provide further evidence for the usefulness of culturing cell lines as spheroids in the process of understanding important cell biological phenomena.

Penetration of substances into tumour tissue. Model studies using saccharides, thymidine and thymidine-5'-triphosphate in cellular spheroids.

In order to achieve a better understanding of factors involved in drug penetration into poorly vascularized tumour tissue, the penetration of some model substances was studied in vitro. Multicellular human tumour spheroids were used as model system. The test substances were [3H]thymidine and [14C]glucose, both of which are capable of passing easily through cell membranes, and [3H]thymidine-5'-triphosphate, [3H]sucrose and [3H]inulin, all of which are unable to pass directly through cell membranes. The penetration of these substances was studied using a dry histological and autoradiographical method preserving the distribution of water-soluble substances. The two thymidine compounds penetrated very efficiently into the spheroids, and their penetration patterns were rather similar. The saccharides differed somewhat in their penetration properties. Glucose had the fastest penetration and inulin the slowest. After 15 min, however, inulin was also found isotropically distributed within the spheroids. Thus, extracellular penetration seemed to be a possible way for a substance to reach the central parts of a spheroid. The differences between the saccharides could be due to some extent to differences in molecular weight and solubility.

Influence of glucose and buffer capacity in the culture medium on growth and pH in spheroids of human thyroid carcinoma and human glioma origin.

pH gradients were measured with microelectrodes in cellular spheroids of human glioma (U-118 MG) and thyroid carcinoma (HTh7) origin. pH decreased outside the spheroids and then continuously decreased when the electrode was moved through the spheroid towards the center. The lowest pH values inside the spheroids were in the range 6.7-6.8 when grown under standard conditions with F10 medium. When medium with stronger buffer capacity (Dulbecco's minimum essential medium or Locke's) was used, the gradients in both types of spheroids were less steep. Less steep pH gradients were also obtained in both types of spheroids when the concentration of glucose was lowered to 0.1 g/liter in the medium. In the case of HTh7 spheroids the low pH inside the spheroids under standard culture conditions seemed toxic because the growth rate increased when the spheroids were cultured under conditions giving higher central pH values (high buffer capacity or low glucose concentration). No such growth-stimulating effects could be seen for the U-118 MG spheroids. The growth rate of both types of spheroids was retarded when they were grown in medium with very high glucose concentration (10 g/liter). The thickness of the viable cell layer increased for HTh7 spheroids when the concentration of glucose was lowered to 0.1 g/liter. A decrease in the thickness of the viable layer of U-118 MG spheroids was observed when they were grown at a high glucose concentration (10 g/liter).

The quantitation of human growth hormone by a radioreceptor assay using an established human cell line.

Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The assay should fulfil established pharmacopoeial requirements for quantitation of hormones. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 X 10(7) cells ml-1 were incubated with 5-20 X 10(-12) M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations.

Demonstration of an extracellular matrix in multicellular tumor spheroids.

Multicellular spheroids of a human glioma cell line (U-118 MG) and a human thyroid cancer cell line ( HTh -7) were analyzed for the presence of extracellular matrix (ECM) using light microscopy, transmission electron microscopy, and indirect immunofluorescence staining for fibronectin, laminin, and collagen. These studies were supplemented by analyses of glycosaminoglycans using autoradiography or chemical methods after metabolic labeling with [35S]sulfate or [3H]glucosamine in conjunction with various extraction procedures. The results showed that both types of spheroids contained an ECM composed of fibronectin, laminin, collagen, and glycosaminoglycans. The organization of the ECM in the spheroids seemed to be similar to that of tumors in vivo. These findings help justify the use of the spheroid system as an in vitro model for the study of biological phenomena of human tumors in vivo. Furthermore, it is concluded that the formation of an ECM in vitro is not confined to normal cells but can be promoted in transformed cells using appropriate culture conditions.

Effects of vinblastine and 5-fluorouracil on human glioma and thyroid cancer cell monolayers and spheroids.

The effects of the two antitumor drugs vinblastine and 5-fluorouracil on the growth of the human tumor cell lines U-118 MG (glioma) and HTh-7 (thyroid cancer) were analyzed. The cells were cultured both as monolayers and as multicellular spheroids and exposed to vinblastine (0.1, 1.0, or 10 micrograms/ml) or 5-fluorouracil (10, 100, or 1000 micrograms/ml) for 15 min, 2 hr, or 24 hr. The drugs induced growth delays of the monolayers and delays in the outgrowth of cells from spheroids which were placed on cell-adhesive surfaces. Cell cultures exposed to sublethal drug doses showed a dose-dependent lag period followed by regrowth at normal growth rates. In all cases with vinblastine exposures, the spheroids seemed more resistant to the drug treatments than did the monolayer cultures. Much smaller differences were obtained after treatments with 5-fluorouracil. The three-dimensional arrangement of cells in spheroids giving rise to, e.g., nutrient and proliferation gradients may, to some extent, be responsible for the increased resistance. The spheroids were especially resistant to short treatments with vinblastine. This was probably due to penetration barriers.

Penetration and binding of vinblastine and 5-fluorouracil in cellular spheroids.

The kinetics of the penetration and binding of the two commonly used antitumour drugs vinblastine and 5-fluorouracil in nonvascularized tumour tissue were studied. Multicellular human tumour spheroids (glioma U-118 MG and thyroid cancer HTh-7) were used as a model system. Radiolabelled drugs were used in all studies. To avoid disturbances in the distribution of unbound drugs a dry histological technique was used in combination with contact autoradiography. In addition, quantitative measurements of the accumulation and binding of the drugs were made. The results showed that vinblastine penetrated the spheroids less efficiently than 5-fluorouracil. Vinblastine required about 2 h to be isotropically distributed within the studied spheroids, while only a few minutes were required for 5-fluorouracil. Vinblastine seemed to be accumulated in the peripheral parts of the spheroids within 15 min. High concentrations of 5-fluorouracil, isotropically distributed in the spheroids, were observed after 2 h of incubation. Significant amounts (about half) of the accumulated drugs resisted gentle washing for 3 X 20 s plus 15 min in fresh medium. The limited penetration of vinblastine correlated well with a previously observed high resistance of spheroids to treatments of short duration with this drug.

Penetration of substances into tumor tissue: a methodological study with microelectrodes and cellular spheroids.

A new method was tested for studies of penetration of substances into tumorlike tissue. The penetration of the ions K+, Cl-, and Ca2+ through several layers of tumor cells was demonstrated by using double barrelled, ion sensitive microelectrodes with extra thin tip diameters. Spheroids consisting of human glioma, U-118 MG, and human thyroid cancer, HTh-7, cells were used as models of tumor tissue. A microelectrode was inserted into the center of a spheroid. Thereafter, the concentration of the test substance was increased in the surrounding medium. The change in concentration inside the spheroid was recorded and the penetration pattern evaluated. All three types of tested ions penetrated easily through the spheroids. The K+ ions penetrated most efficiently, and the Ca2+ ions showed the slowest penetration. The Ca2+ ions penetrated somewhat more slowly in the U-118 MG spheroids (which had rather small extracellular spaces) than in the HTh-7 spheroids (which had larger extracellular spaces). Ion sensitive electrodes, which are easily available, were used in this study only to demonstrate the principle. We hope that the method described can be used for penetration studies of various substances. For example, all substances that can be detected by enzyme microelectrodes could be studied. The main advantage of the method is that the complete penetration pattern can be studied as a function of time in individual spheroids. Previously described methods require histological procedures for each analyzed penetration time.

Methodological aspects of microelectrode measurements in cellular spheroids.

Different types of oxygen microelectrodes have been tested in measurements on cellular spheroids. The shape of the oxygen gradients varied strongly depending on size and type of the spheroids. No significant differences in the results were obtained when different types of electrodes were applied. All measurements were made in a perfusion chamber. The shape of the gradients did not vary with time in the perfusion chamber. The reproducibility was found good in repeated measurements using the same spheroid. No mechanical or chemical disturbances were seen during the penetration of the spheroids. Changes in the medium flow rate through the chamber did not drastically change the shape of the oxygen gradients. Almost no convection could be seen at the bottom of the chamber close to the spheroids. The composition of the medium was found to be of importance. Lock's solution containing glucose was found to be satisfactory. The potential signals in the double barrel electrodes allowed an accurate determination of the position when the electrode hit the spheroid surface. The information gained from microelectrode measurements in spheroids might be valuable for the understanding of effects of new tumor treatment modalities in which hypoxic cell sensitizers or high LET radiation are utilized.

A method to measure the radio and chemosensitivity of human spheroids.

A method based on the spontaneous outgrowth of cells from spheroids was tested. Different outgrowth patterns were seen depending on the types of spheroids and on the radiation or drug doses. The method allowed dose-effect relations to be determined. Spheroid survival was defined as when the outgrowing monolayers contained at least thousand cells within five weeks. The method was used as an alternative to cloning of isolated single cells. The glioma and osteosarcoma spheroids could not be disintegrated to single cell suspensions since they resisted enzymatic and mechanical treatments for cell separation. Detection of differences in radio and chemosensitivity between different types of spheroids of human origin might be valuable for the understanding of the large variations in therapeutical response often seen between different types of tumors.

Effects of interferon on growth rate and radiation sensitivity of cultured, human glioma cells.

The influence of interferon (IFN) on the growth rate and on the radiation sensitivity of 2 human tumour cell lines was investigated. The 2 glioma cell lines (U-118 MG and U-251 MG) were continuously exposed to IFN (100 U/ml) in the culture medium. Irradiation (3 Gy) was performed either on the first day of IF treatment or on day 14 of IFN treatment. The growth delay induced by the treatments was analysed. The results indicated that IFN had an anti-proliferative effect on the 2 cell lines. However, this effect declined during the treatment and after 2 to 3 weeks of continuous IFN treatment, both cell lines had re-established their original growth rate. IFN did not seem to affect the sensitivity of the cells to radiation. Only an additive effect could be observed.

Penetration of substances into tumor tissue--a methodological study on cellular spheroids.

The penetration of [3H]thymidine, [3H]D-leucine, [125I]albumin, and the drugs [3H]5-fluorouracil and [3H]vinblastine into human glioma spheroids (in vitro tumor models) was studied by a method based on rapid freezing, freeze drying, vapor fixation, wax embedding, dry sectioning, and contact autoradiography. No significant disturbances in the distribution of water soluble substances were observed. Thymidine and D-leucine penetrated the whole spheroids relatively fast, whereas albumin showed reduced penetration. The concentration of albumin was highest at the periphery of the spheroids, but only smaller amounts were detected in the deeper regions. A significant difference between the penetration patterns of the drugs studied was also observed. Fluorouracil penetrated rather freely, but the penetration of vinblastine was limited.