An EORTC-ECSG phase I study of LU 79553 administered every 21 or 42 days in patients with solid tumours.

A single-agent dose-escalating phase I and pharmacokinetic study on the naphthalamide agent, LU 79553, was performed to determine its safety profile, maximum tolerated dose (MTD) and recommended dose for phase II studies. LU 79553 was given intravenously (i.v.) every 3 weeks to patients with advanced solid cancers (an extended cohort of patients also received the drug every 6 weeks). 59 patients were enrolled into the study (50 patients in the 3-weekly schedule and 9 patients in the 6-weekly schedule). Dose levels studied ranged from 10 mg/m(2) to 160 mg/m(2). Neuro-muscular toxicity was identified as the dose-limiting toxicity (DLT). This muscular toxicity was observed after administrating total doses of 160-450 mg/m(2) (median 330 mg/m(2)). Non-DLTs consisted of diarrhoea, nausea and vomiting, fatigue and local venous phlebitis. The major haematological toxicities observed were anaemia and neutropenia (and were mainly observed at the two highest dose levels). The proposed dose for phase II studies using the 3-weekly regimen is 100 mg/m(2)/course (60 min infusion in 500 ml normal saline), but a close clinical follow-up of the patients for neuromuscular toxicity is mandatory. Prolongation of the treatment interval to 6 weeks, based upon the long half-life of the drug in the plasma and tissue, observed during this study, seemed not to be feasible in this heavily pretreated group of patients.

Visualization of percutaneous 3H-estradiol and 3H-norethindrone acetate transport across human epidermis as a function of time.

Developing transdermal therapeutic systems for estradiol and norethindrone acetate raised questions about the steroids penetration pathway across and retention in the skin. This paper describes the distribution of 3H-estradiol and 3H-norethindrone acetate in human stratum corneum after topical application to dermatomed skin in vitro. The study involved (a) permeation experiments to determine the steroid flux, (b) autoradiographical visualization of the steroid distribution in the same skin samples, and (c) a correlation between flux and skin distribution in time. On correlating the steroid flux with intraepidermal steroid distribution, it was concluded that both permeants were bound in the skin tissue. The steroids were preferentially located in or close to the intercellular lipids of the stratum corneum, indicating that both transport and binding occurred via this domain of the stratum corneum. This study demonstrated the importance of correlating drug flux with intraepidermal drug distribution as a function of time.

Reflection contrast microscopy for high resolution detection of (3)H-estradiol in ultrathin sections of human stratum corneum.

A single autoradiographical method for light and electron microscopy (LM and EM) is presented. Human skin, containing (3)H-estradiol ((3)H-E2) after an in vitro permeation experiment, was processed via a non-extractive tissue preparation protocol, comprising cryo-fixation, freeze-drying, osmium tetroxide vapor fixation, and Spurr resin embedding. Semithin sections were processed for LM autoradiography, while ultrathin sections were processed both for high-resolution LM and EM autoradiography. The autoradiographs were visualized by bright-field microscopy (BFM), reflection contrast microscopy (RCM), and transmission electron microscopy to evaluate the potentials of RCM visualization in high-resolution LM autoradiography. RCM visualization of ultrathin vs. semithin resin sections showed an improved stratum corneum morphology. Histological staining was superfluous. The localization of (3)H-E2 in human stratum corneum using high-resolution LM autoradiography and RCM was as accurate as with high-resolution EM autoradiography.

Validation of freeze-drying to visualize percutaneous 3H-estradiol transport: the influence of skin hydration on the efficacy of the method.

A study was made of the validity of freeze-drying to visualize the distribution of 3H-estradiol in human stratum corneum after topical application of a dry dose, a patch or a buffer solution. Each of these donor formulations was applied to human dermatomed skin for 24 h using Franz permeation cells. Subsequently, small pieces of skin were subjected to cryofixation, freeze-drying, osmium tetroxide vapor fixation, Spurr resin embedding and electron microscopic autoradiography. Stratum corneum from dry dose and patch application experiments was well preserved by freeze-drying, allowing an accurate localization of 3H-estradiol. In contrast, stratum corneum from buffer solution experiments suffered from cryofixation artifacts due to excessive hydration of the skin. The corresponding autoradiographs showed strong redistribution of 3H-estradiol. Thus, the visualization method under investigation has its limitations regarding the hydration level of the skin.

Development of an optimal protocol for the ultrastructural examination of skin by transmission electron microscopy.

The intercellular lipid bilayers of the stratum corneum provide the permeability barrier of the skin. To perform an electron microscopical examination of the ultrastructure of these bilayers, ruthenium tetroxide fixation is required. In this study an optimal fixation protocol was developed and selected upon comparing seven different fixation procedures, using glutaraldehyde (GA) and the postfixatives ruthenium red, osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) in combination with potassium ferrocyanide (K4Fe(CN)6) and potassium ferricyanide (K3Fe(CN)6). Instead of fixing skin with either OsO4 or RuO4, these two fixatives were combined in one protocol. In addition, the use of RuRed was introduced and the influence of both K4Fe(CN)6 and K3Fe(CN)6 in combination with RuO4 were examined. Furthermore, we compared two dehydration solvents, methanol and acetone. The most satisfying results were obtained when the skin was prefixed in GA and postfixed in OsO4 and RuO4 with K3Fe(CN)6, i.e. with Fe in its highest oxidation state (Fe3+). No differences were observed between dehydration in methanol and acetone.

Visualization of diffusion pathways across the stratum corneum of native and in-vitro-reconstructed epidermis by confocal laser scanning microscopy.

Confocal laser scanning microscopy is a technique that permits the direct visualization in unfixed material of diffusion pathways and the cellular distribution of fluorescent markers after topical applications. This approach, in which the tissue specimen is optically sectioned, allows the study of changes in distribution pattern of applied compounds depending on the vehicle, time and depth without the interference of chemical alterations induced by most of the current techniques used for such studies. Using this technique the permeability properties of in-vitro-reconstructed epidermis were compared with those of the native counterpart. The epidermis was reconstructed by culturing human adult keratinocytes at the air-liquid interface either on fibroblast-populated collagen or on de-epidermized dermis. A fluorescent probe--Nile red (NR)--was applied in three different vehicles--polyethylene glycol (PEG) with a molecule mass of 400 (Da), propylene glycol (PG) and dimethyl sulphoxide (DMSO)--which perturb the SC barrier function to different extents. When NR was applied in PEG and PG on native epidermis, the amount of NR penetrating into and through the SC was very low, but was markedly increased when NR was applied in DMSO. Unlike native epidermis, the reconstructed epidermis allowed rapid NR penetration after the application in any of the solvents used. Furthermore, NR applied on reconstructed epidermis, was distributed quite homogeneously between the cellular and the intercellular spaces throughout the SC, suggesting that not only intercellular lipid structures but also the properties of the cornified envelopes differed markedly from those found in native epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)

MAb U36, a novel monoclonal antibody successful in immunotargeting of squamous cell carcinoma of the head and neck.

Using viable cells of a human head and neck squamous cell carcinoma (HNSCC) cell line as immunogen, we developed monoclonal antibody (MAb) U36. Immunohistochemical examination revealed distinct surface labeling of MAb U36 with normal human squamous epithelium and squamous cell carcinomas of distinct sites of origin; head and neck, lung, esophagus, cervix, and epidermis. MAb U36 shows high affinity binding (affinity constant, 3.5 x 10(10)/M) with a cell surface antigen expressed by in vitro cultured HNSCC cell lines. Similarity of the reactivity profiles of MAb U36 and MAb E48, currently the most promising antibody described for specific targeting of HNSCC in patients, warranted further comparison of these MAbs. MAb U36 recognizes a M(r) 200,000 antigen, which is different from the MAb E48 defined antigen. Furthermore, comparison of immunohistochemical staining patterns of MAb U36 and MAb E48 on a broad panel of primary HNSCC sections revealed more extensive staining for MAb U36: more tumors showed reactivity with MAb U36 and more tumor cells per tumor showed positive reaction, and staining was found to be more intense. MAb U36 does not show cross-reactivity with mouse, rat, pig, sheep, or bovine tongue epithelium. As a first approach to evaluate the suitability of MAb U36 for tumor targeting in vivo, radiolabeled MAb U36 was administered to athymic nude mice bearing human HNSCC xenografts on both flanks. Selective tumor accumulation of the radioimmunoconjugate was observed. Mean tumor uptake (in percent injected dose/g wet-weight of tissue) of MAb U36 at days 1, 2, 3, 5, 7, and 12 was 15.1, 17.9, 24.0, 21.0, 25.8, and 16.0%, respectively. The tumor to blood ratio at day 1 was 0.9 and increased up to 3.8 at day 12. The tumor uptake at day 12 was at least 10 times higher when compared to other tissues. The corollary of these findings is that MAb U36 harbors high potential for specific targeting of HNSCC.

Radioimmunotherapy of human head and neck squamous cell carcinoma xenografts with 131I-labelled monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 reacts with a 22 kD antigen exclusively expressed in squamous and transitional epithelia and their neoplastic counterparts. Radiolabelled with 99mTc, MAb E48 is capable of targeting metastatic and recurrent disease in patients with head and neck cancer. In this study, the capacity of 131I-labelled MAb E48 to eradicate xenografts of human squamous cell carcinoma of the head and neck (HNSCC) in nude mice was examined. Experimental groups received a single i.v. bolus injection of 400 microCi MAb E48 IgG (number of mice (n = 6, number of tumours (t) = 9) or 800 microCi MAb E48 IgG (n) = 5,t = 7), whereas control groups received either diluent (n = 3,t = 5), unlabelled MAb E48 IgG (n = 4,t = 5) or 800 microCi 131I-labelled isotype-matched control MAb (n = 6,t = 9). A 4.1-fold increase in the median tumour volume doubling time and regression of two out of ten tumours (20%) was observed in mice treated with 400 microCi. In mice treated with 800 microCi. In mice treated with 800 microCi, two out of seven tumours (29%) showed complete remission without regrowth during follow-up (greater than 3 months). Median tumour volume doubling time in the remaining five tumours was increased 7.8-fold. No antitumour effects were observed in mice injected with diluent, unlabelled MAb E48 or 131I-labelled control MAb. In the same xenograft model, chemotherapy with doxorubicin, 5-fluorouracil, cisplatin, bleomycin, methotrexate or 2',2'-difluorodeoxycytidine yielded a less profound effect on tumour volume doubling time. Increases in tumour volume doubling time with these chemotherapeutic agents were 4, 2.2, 2.1, 1.7, 0, and 2.6 respectively. Moreover, no cures were observed with any of these chemotherapeutic agents. From the tissue distribution of 800 microCi MAb E48, the absorbed cumulative radiation doses of tumour and various organs were calculated using the trapezoid integration method for the area under the curve. To tumour xenografts, 12,170 cGy was delivered, blood received 2,984 cGy, whereas in every other tissue the accumulated dose was less than 6% of the dose delivered to tumour. These data, describing the first radiolabelled MAb with therapeutic efficacy against HNSCC, suggest radioimmunotherapy with MAb E48 to be a potential therapeutic modality for the treatment of head and neck cancer.

Potential for targeting head and neck squamous cell carcinoma with monoclonal antibody K984.

In previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratios, as based on the biodistribution data, were at least ten times higher in case of the specific mAb K984 when compared to another non-specific, isotype-matched control antibody. mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targeting.

Detection of squamous cell carcinoma xenografts in nude mice by radiolabeled monoclonal antibody E48.

We recently produced the monoclonal antibody E48 as a specific reagent for squamous cell carcinomas. In our ongoing investigations to use E48 for clinical tumor detection and therapy, fundamental aspects of the antigen have to be elucidated and practical applications of the antibody have to be tested in a preclinical model. Immunoelectron-microscopic studies localized the E48 antigen along the cell surface and in between desmosomes, suggesting that the antigen serves as an adhesion molecule. To evaluate the usefulness of E48 for radioimmunodetection of neck node metastases, nodes from 20 neck dissection specimens were tested. A strong reactivity was observed. Furthermore, F(ab')2 fragments of E48 were compared with the complete IgG E48 for selective tumor detection in an animal model. It was demonstrated that E48 F(ab')2 fragments localize faster and reach higher tumor-nontumor ratios than the whole molecule.

Evidence for a role of the monoclonal antibody E48 defined antigen in cell-cell adhesion in squamous epithelia and head and neck squamous cell carcinoma.

Monoclonal antibody (MAb) E48 recognizes a 20- to 22-kDa antigen expressed by human squamous and transitional epithelia and their neoplastic counterparts. Histochemical examination of these tissues revealed distinct surface labeling with MAb E48. To investigate the subcellular localization of the E48 antigen we have performed electron microscopical analysis. In cells of normal oral mucosa, the E48 antigen was expressed on the plasmalemma, particularly associated with desmosomes, suggesting involvement of the E48 antigen in intercellular adhesion. Furthermore, the level of expression of the E48 antigen appeared to be influenced by the cellular organization. In squamous cell carcinoma (SCC) cell lines grown in vitro as subconfluent monolayer cultures, the E48 antigen expression was low. However, E48 antigen expression increased when SCC cells were grown to confluency. E48 antigen expression was similarly high when SCC cell lines were cultured under conditions promoting three-dimensional growth either as colonies within floating collagen gels or as xenograft in tumor-bearing nude mice. Further evidence for the involvement of the E48 antigen in cell-cell adhesion was found when SCC cells were grown within collagen gels in the presence of MAb E48: no spherical colonies were formed, but cells grew out to colonies composed of single cells. Moreover, in this culture system the percentage of SCC cells growing out to colonies was decreased by the presence of MAb E48. These findings indicate that the E48 antigen is involved in the structural organization of squamous tissue and might have a role in intercellular adhesion.

Ischemia and reperfusion induced multilamellar vesicles in isolated rabbit hearts: time correlation between morphometric data and metabolic alterations.

In normoxic hearts a limited number of multilamellar vesicles was found in both endothelial cells and myocytes. The total number of multilamellar vesicles observed in myocytes, particularly those extruded from mitochondria, significantly increased in hearts rendered ischemic for at least 60 mins. The number of multilamellar vesicles extruded from sarcolemma was increased in hearts reperfused after this period of ischemia. The number of multilamellar vesicles in or adjacent to lipid droplets was independent of the duration of ischemia. Multilamellar vesicles were similar in size and periodicity of the lamellae. It is proposed that the number of multilamellar vesicles can be used to quantitate ischemic membrane injury. The formation of multilamellar vesicles was significantly related in time to (a) the accumulation of arachidonic acid and total fatty acids; (b) a decrease in the tissue content of ATP and (c) the release of lactate dehydrogenase (LDH). No significant alterations in the total tissue content of triacylglycerols and phospholipids were detected. The amount of arachidonic acid accumulated in the hearts reflects the degradation of only a minor fraction of the phospholipid pool. Assuming a close relationship between phospholipid degradation, induction of multilamellar vesicles and loss of cellular integrity, the present findings might indicate that the loss of a small part of phospholipids might have serious pathophysiological consequences, as indicated by the morphological changes in cellular membranes and the release of cytoplasmic macromolecules.

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.

Dual effect of tannic acid on the preservation and ultrastructure of phosphatidyl choline vesicles.

The use of tannic acid has been proposed to improve the preservation of phospholipids in tissues. We investigated the effects of tannic acid on the preservation of small unilamellar vesicles, prepared from sonicated aqueous suspensions of phospholipids. With cryo-electron microscopy it is demonstrated that small unilamellar vesicles are formed after sonication of the phospholipid suspensions. Fixation of vesicles without tannic acid results in extraction of the phospholipids during dehydration and embedding. Fixation of vesicles containing phosphatidyl choline with tannic acid, with or without glutaraldehyde, results in a fast (within a second) aggregation of the vesicles and the resulting sediment can be dehydrated and embedded when a postfixation in osmium tetroxide is carried out. Small unilamellar vesicles fixed in this way are retrieved in thin sections as multilamellar vesicles with a periodicity of about 5 nm for dimyristoylphosphatidyl choline and about 6 nm for dioleoylphosphatidyl choline. By using 14C-phosphatidyl choline it was also demonstrated that tannic acid prevents to a large extent the extraction of phosphatidyl choline during fixation, dehydration and embedding. This dual effect of tannic acid on phosphatidyl choline, aggregation and fixation, should be considered when using tannic acid in tissue preparation.

Degradation of phospholipids and triacylglycerol, and accumulation of fatty acids in anoxic myocardial tissue, disrupted by freeze-thawing.

The degradation of lipids by endogenous hydrolytic activity has been studied in rat cardiac tissue deliberately damaged by freezing and thawing prior to storage under anoxic conditions. Aliquots of the freeze-thawed material were kept at 37 degrees C under an atmosphere of N2 up to 120 minutes. Triacylglycerol was hydrolyzed at a rate of 0.14 mumol fatty acids per minute per gram dry weight of tissue. Hydrolysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was associated with proportional production of lyso PC and lyso PE, respectively. This finding indicates that the activity of lysophospholipase is negligible in autolyzing cardiac tissue. The rate of hydrolysis of PC and PE was found to be 0.10 and 0.06 mumol per minute per gram dry weight of tissue. The observation that lyso PC and lyso PE mainly contained saturated and mono-unsaturated fatty acids indicates that phospholipase A2 rather than A1 is active in autolyzing cardiac tissue. The accumulation of fatty acids corresponded with the loss of triacylglycerol and phospholipids from the tissue during 120 minutes of autolysis.