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Biomedical subjects

E R Huehns

Publications and source records attributed to E R Huehns.

At least 19 recordsLinked to original sources
PubMed1992

Use of photosensitive, antibody directed liposomes to destroy target populations of cells in bone marrow: a potential purging method for autologous bone marrow transplantation.

Liposomes containing the photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) were coupled to polyclonal sheep anti-mouse-Ig antibody and bound to cells coated with specific mouse monoclonal antibody. When illuminated with red light, the AlSPc in the liposomes was activated to produce singlet oxygen and the antibody and liposome targeted cells were destroyed. DW-BCL cells (an Epstein Barr virus immortalised B-cell line) were targeted with an anti-B-cell antibody (8A) and killed specifically, both alone and in the presence of bone marrow mononuclear cells (BM-cells), without phototoxic effects on the untargeted bone marrow CFU-GM progenitor cells. The presence of an excess of non-target cells did not interfere with antibody and liposome binding, or light access to target cells. Similar results were obtained with T-lymphocytes as target cells using anti-CD3 antibody. Specific targeting to the B-cells was demonstrated in the cell mixtures by use of fluorescent microscopy combined with a sensitive technique to detect low levels of AlSPc fluorescence, a cooled charge couple device (CCD) camera. This was also able to show low levels of non-specific background binding of AlSPc to BM-cells and a small population of cells that took up AlSPc in the absence of antibody. The latter were shown to be monocytes by flow cytometry.

Bone Marrow↗
PubMed1991

Comparison of the subacute toxicity and efficacy of 3-hydroxypyridin-4-one iron chelators in overloaded and nonoverloaded mice.

Five orally effective iron chelators of the 3-hydroxypyridin-4-one series have been administered intraperitoneally to iron-overloaded and nonoverloaded male mice at a dose of 200 mg/kg/24 h for a total of 60 days to investigate the effect on iron loading and toxicity. There was a significant reduction in hepatic iron at the end of the study in the iron-overloaded mice with all compounds studied using chemical iron quantitation (P less than .001) and with Perls' stain (P less than .01). Liver iron removal with the hydroxypyridinones ranged from 37% with CP20 to 63% with CP51, compared with 46% removal for desferrioxamine (DFO). There was no significant reduction in splenic or cardiac iron with any chelator. There were no deaths in iron-overloaded animals receiving any of the hydroxypyridin-4-ones, but significantly more deaths in the nonoverloaded groups as a whole (P less than .03). No weight loss was observed with any chelator. Significant reductions in hemoglobin and white cell count were observed with CP20(L1). No histologic abnormalities of kidney, spleen, bone marrow, or stifle joints were observed. Intracytoplasmic inclusion bodies were observed in the centrilobular hepatocytes of animals administered each of the hydroxypyridin-4-ones, while the DFO-treated and control groups showed no such changes.

Animals↗
PubMed1991

Congenital spherocytosis, B19 parvovirus infection and inherited interstitial deletion of the short arm of chromosome 8.

We report two siblings with congenital spherocytosis, multiple phenotypic abnormalities and an inherited interstitial deletion of the short arm of chromosome 8 (8p). The propositus came to our attention with acute bone marrow hypoplasia secondary to B19 parvovirus infection. The bone marrow trephine biopsy appearances of intranuclear eosinophilic degeneration in the erythroblasts may be pathognomonic of B19 parvovirus induced acute bone marrow aplasia. The presence of B19 parvovirus DNA was demonstrated in erythroblasts by in situ hybridization. Chromosome analysis of peripheral blood lymphocytes from both siblings showed an interstitial deletion of the short arm of chromosome 8, del (8) (p11p21). This abnormal chromosome was inherited from their mother, who showed this deletion as well as a small fragment representing the deleted 8p chromosome portion, del (8) (p11p21), +f. Centromeric material from chromosome 8 was detected in this chromosome fragment by in situ hybridization using an alpha satellite probe (pJM 128), but not by C banding. Chromosome analysis of skin fibroblasts from the mother and a third sibling with a similar karyotype showed the deleted fragment in over 80% of cells. Cells in which the fragment was absent exhibited the deleted 8p, suggesting there was no mosaicism. The mother and the third sibling were phenotypically normal without spherocytosis. A fourth sibling and the father were normal. The chromosome abnormality was not observed in five of the mother's siblings, suggesting that it arose de novo in the mother. Our findings strongly support a locus for congenital spherocytosis on the short arm of chromosome 8. The frequency of defects at this locus is unknown.

Adolescent↗
PubMed1990

Relative oral efficacy and acute toxicity of hydroxypyridin-4-one iron chelators in mice.

The relationship between the oral efficacy and the acute toxicity of hydroxypyridin-4-one iron chelators has been investigated to clarify structure-function relationships of these compounds in vivo and to identify compounds with the maximum therapeutic safety margin. By comparing 59Fe excretion following oral or intraperitoneal administration of increasing doses of each chelator to iron-overloaded mice, the most effective compounds have been identified. These have partition coefficients (Kpart) above 0.3 in the iron-free form with a trend of increasing oral efficacy with increasing Kpart values (r = .6). However, this is achieved at a cost of increasing acute toxicity, as shown by a linear correlation between 59Fe excretion increase per unit dose and 1/LD50 (r = .83). A sharp increase in the LD50 values is observed for compounds with Kpart values above 1.0, suggesting that such compounds are unlikely to possess a sufficient therapeutic safety margin. Below a Kpart of 1.0, acute toxicity is relatively independent of lipid solubility. All the compounds are less toxic by the oral route than by the intraperitoneal route, although iron excretion is not significantly different by these two routes. At least five compounds (CP51, CP94, CP93, CP96, and CP21) are more effective orally than the same dose of intraperitoneal desferrioxamine (DFO) (P less than or equal to .02) or orally administered L1(CP20) (P less than or equal to .02).

Administration, Oral↗
PubMed1989

Enhanced fluorescence in indirect immunophenotyping by the use of fluorescent liposomes.

Small unilamellar liposomes were optimised for cell phenotyping by indirect immunofluorescence. This involved selection and covalent attachment to the liposome of a polyspecific ligand for cell-bound antibody. For this purpose sheep anti-mouse antibody was preferred to protein A because of its ability to attach to cell-bound IgG1 as well as IgG2 at physiological pH. The maximally fluorescent concentration of encapsulated carboxyfluorescein was determined to be 20 mM and liposomes thus comprised gave up to a nine-fold increase in mean cell fluorescence when compared with sheep anti-mouse antibody conjugated to fluorescein isothiocyanate. There was no parallel increase in background fluorescence. Liposomes retained their targeting and fluorescence properties after 3 months storage. They could be sterilised and were as versatile in use as FITC-antibody conjugates.

Animals↗
PubMed1989

The development of iron chelating drugs.

Studies over the past few years have shown that it is possible to develop iron chelating agents that are active when given by mouth. Such compounds need to have a high binding constant for Fe(III) and an intermediate water and lipid solubility of both the unliganded compound and the iron complex with a Kpart of 0.2-1 for the free ligand. Hexadentate ligands would be preferable to bidentate compounds but no suitable compounds are available. In order to evaluate such compounds, simple cellular and animal screening models have been set up in a number of laboratories, and the potential of a new compound can be determined in a few weeks. Several groups have produced candidate compounds which are in various stages of development. DF is the established iron chelating drug, and the production of an orally active pro-drug must be high on the list of further developments, although this approach has so far not produced any useful compounds. It would appear that, at present, none of the other available hydroxamates will be sufficiently orally active or non-toxic to replace DF. Similarly, none of the available catecholates is promising enough to warrant further development at present. Among the amino carboxylates, although specificity may be a problem, the ester derivatives of HBED appear promising but have not yet been fully evaluated; this needs to be done before this group is discarded. Among the aryl hydrazones, PIH has reached the stage where it has been given to humans, but it may not be sufficiently active to be clinically useful. In this group there are several further compounds under development. PIB and a number of other derivatives need careful investigation before they are discarded, while pyridoxal-2-pyrimidyl-ethoxycarbonyl methbromide (PPEM) is in the early stages of animal testing and appears quite promising. Too little is known about the more recently synthesized hexadentate compounds based on the pyridoxal moieties, such as PLED, to judge whether this is a promising approach. Unfortunately, the naturally occurring siderophore, desferrithiocin, has proved too toxic for further development. Finally, a number of hydroxypyridin-4-ones have been synthesized and there are several that appear to be promising. CP20 (L1), the methyl derivative, has been given to humans, while at least two compounds with greater activity and relatively lower animal toxicity are close to being introduced. This group of compounds lends itself to the synthesis of a large number of derivatives with defined properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

The toxic effects of desferrioxamine.

DF has a low general toxicity, perhaps because of its low lipid solubility, Kpart 0.01 (Porter et al, 1988b). This feature of the molecule may prevent it from penetrating most cells of the body. It appears that there may be a specific mechanism of uptake of the drug by hepatocytes (Porter et al, 1987), making the iron in these cells available for excretion via the bile, while the iron excreted in the urine may all come from extracellular chelation, particularly when iron leaves the reticuloendothelial cells (Hershko et al, 1978). On this hypothesis, cellular toxicity occurs only when DF penetrates sensitive cells in sufficient amounts so that some free DF remains after all the available iron in such cells has been chelated. Such a hypothesis accounts for the protection of cells by iron overload and therefore the greater sensitivity of unloaded patients. The retina and central nervous system are further protected by the blood-retinal or blood-brain barrier, and increased penetration of this barrier, mediated by high peak levels of DF, by drugs or other diseases would lead to the retinal or neurotoxic effects seen. In the ear, high levels of unliganded DF for a period of time may be necessary to cause deafness. Thus the very property that prevents its oral activity may be part of the reason for the low toxicity of DF. The severe toxic effects on vision, hearing and growth are all more likely at higher doses of DF and there appears to be partial protection against them by iron overload. These two conclusions have to be taken into account when deciding on the appropriate dosage for each patient. With care, the dosage can be adjusted to remove enough iron to prevent iron accumulation and therefore its toxic effects, whilst keeping doses low enough to prevent DF from being toxic itself. It appears that even in very iron-overloaded patients dosages higher than 125 mg kg-1 day-1 may cause visual disturbances and should be avoided. In patients on renal dialysis with aluminium toxicity great care is needed to avoid retinal toxicity even with dosages as low as 50 mg kg-1 day-1, although the drug should not be withheld if clinically indicated. The administration of DF to renal dialysis patients is described by Pogglitsch et al (1981, 1983), Pacitti et al (1983), Ihle et al (1986) and Molitoris et al (1987). DF should not be given to patients unless there is a clearly established clinical indication.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Specific targeting and toxicity of sulphonated aluminium phthalocyanine photosensitised liposomes directed to cells by monoclonal antibody in vitro.

A partially purified fraction of the water soluble photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) was encapsulated in liposomes which were then linked to a targeting monoclonal antibody 791T/36 using a heterobifunctional linking agent. The photocytotoxic effects of the liposomes were determined on two cell lines bearing an antigen with which the targeting antibody binds: 791T, an osteosarcoma and C170, a colorectal carcinoma; and a control cell line not bearing the antigen; DW-BCL, an Epstein-Barr virus immortalised B-cell line. Antibody dependent cytotoxicity was observed in 791T and C170 cells and was proportional to the number of antigens on the cells, the AlSPc concentration and the time of exposure to activating red light. No significant toxicity was seen using untargeted liposomes, control cells or free AlSPc fraction under similar conditions. Targeted cells and controls kept in the dark also showed no significant toxicity. A possible mechanism of action is postulated and simple adaptations which demonstrate the versatility of the model are discussed. Some suggestions as to the clinical situations to which this system might be applied in the form of photodynamic therapy (PDT) are made.

Antibodies, Monoclonal↗
PubMed1989

Desferrioxamine ototoxicity: evaluation of risk factors in thalassaemic patients and guidelines for safe dosage.

Forty-seven patients with thalassaemia have been studied to define risk factors for development of sensorineural hearing loss, and to establish guidelines for safe chelation. Sensorineural hearing loss was only present in patients who had previously received desferrioxamine (DFO). The two most significant risk factors were the maximum dose of DFO previously received (P less than 0.01), and a serum ferritin of less than 2000 micrograms/l at that time (P less than 0.001). A therapeutic index obtained from the ratio of the mean daily dose of DFO mg/kg divided by the serum ferritin identifies patients with a ratio of greater than 0.025 as at risk of sensorineural hearing loss (P less than 0.001) and can be used as a guideline for safe DFO dosage. Follow-up audiometry of the affected patients over a 2-year period indicated that adjustment of the dose to a therapeutic index of less than 0.025 resulted in the stabilization of hearing loss in seven patients and improvement in two.

Adolescent↗
PubMed1988

Differential phthalocyanine photosensitization of acute myeloblastic leukaemia progenitor cells: a potential purging technique for autologous bone marrow transplantation.

The potential value of sulphonated aluminium phthalocyanine (AISPc) as a purging agent for bone marrow autografts in acute myeloblastic leukaemia (AML) has been studied using in vitro clonogenic assays for normal (GM-CFC) and leukaemic (AML-CFC) progenitor cells. In nine out of 13 cases, the leukaemic blasts were found to be highly sensitive to AISPc. In six of the sensitive cases clonogenic assays revealed that only 2 +/- 1% of AML progenitor cells survived AISPc treatment under conditions which permitted a GM-CFC recovery of 60 +/- 11%. AISPc photosensitization was also shown to selectively eliminate the leukaemic cell line K562 from an in vitro model of minimal residual disease. Thus photosensitization using AISPc may be an effective method of purging marrow autografts in some cases of AML. Evaluation of the sensitivity of AML clonogenic cells at diagnosis may identify those patients in whom AISPc photo-purging may be of benefit at the time of an autologous bone marrow transplant.

Bone Marrow Transplantation↗
PubMed1988

Selection of hydroxypyridin-4-ones for the treatment of iron overload using in vitro and in vivo models.

The hydroxypyridin-4-one group of iron chelators show promise as potential compounds for the treatment of iron overload by the oral route. In the search for the compounds best suited for long term clinical use, a balance has to be struck between the desire to mobilise the maximum amount of iron and the wish to minimise the potential toxicity of such compounds. In this article we review the approach we have used to evaluate which of the hydroxypyridinones have the properties best suited for further development prior to clinical trials in man. The diversity of a number of closely related compounds substituted on the ring nitrogen have allowed us to study the properties of chelators responsible for cellular mobilisation of iron(III), as well as those which may contribute to their toxicity. The primary hepatocyte culture model has facilitated the investigation of the contribution of their iron binding constant, as well as the critical importance of their relative lipid solubility to both cellular iron mobilisation and toxicity. Similarly studies in mice have confirmed that the factors affecting cellular iron release also control iron excretion in whole animals. Further we have demonstrated that the acute toxicity of this group of compounds is closely linked to the size of the available iron pool.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant.

A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4-ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2-ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.

Animals↗
PubMed1988

Uptake of antibody directed cytotoxic liposomes by CD3 on human T cells.

Using polyclonal human T cells and anti-CD3 monoclonal antibodies we have shown that small unilamellar liposomes covalently coupled with protein A become bound to T cells and not to B cells and that the binding was a specific liposome-antibody-receptor interaction. Intracellular delivery of liposome contents was demonstrated by the use of encapsulated carboxyfluorescein and flow cytometry and the transfer of membrane-bound liposomal carboxyfluorescein was virtually complete in 30 min. Liposomes containing methotrexate inhibited the growth of PHA-stimulated peripheral blood lymphocytes by 90%, after 48 h incubation. Potential applications are proposed in the study of the behaviour of surface membrane components and in T cell depletion and purging of bone marrow.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Production and characterisation of a human monoclonal antibody to cytomegalovirus and its use in an early nuclear fluorescence assay.

A human monoclonal antibody to cytomegalovirus (CMV) was produced by transforming peripheral blood mononuclear cells of a patient with recent CMV infection. It is directed against a late antigen located in the nucleus of CMV infected fibroblasts at 24-72 hours postinfection and immuneprecipitates 65K and 48K proteins from 35S-labelled CMV infected cells. Results of its use in an early nuclear fluorescence assay for rapid diagnosis are presented.

Adult↗