Bengt S. Larsson

Studies on the mechanism of drug-binding to melanin

Abstract The binding to melanin of chlorpromazine, chloroquine, paraquat and Ni 2+ has been studied in vitro with pigment from beef eyes. The results showed a marked influence of the ionic environment on the ability of the organic substances to bind to melanin, indicating that electrostatic forces between the cationic forms of the substances and anionic sites on the melanin polymer (presumably carboxyl groups) are important for the complex formation. An analysis of the binding by the method of Scatchard showed that more than one binding class must be implicated in the binding of both the organic substances and Nr 2+ to melanin. Several concordances were found for the data of the paraquat- and Ni 2+ -binding, indicating a dominant influence of electrostatic forces for the melanin-binding of paraquat. However, several indications were found that non-electrostatic contributions must be added to form the binding-sites for chlorpromazine and chloroquine. It is possible that such contributions may be provided by van der Waals forces occurring at the conjunctions of the aromatic rings in the substances and the aromatic indole-nuclei of the melanin. Experiments with chlorpromazine indicated that the positive ion radical of the substance had a very high melanin-affinity. It is suggested that melanin may be able to oxidize chlorpromazine to a positive ion radical, explaining the firm binding of the substance to melanin and the evidence in the literature favouring this possibility are discussed.

Autoradiography of [14C]paraquat or [14C]diquat in frogs and mice: Accumulation in neuromelanin

The herbicide paraquat has been suggested as a causative agent for Parkinsons disease because of its structural similarity to a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which may induce a parkinsonism-like condition. MPTP as well as its metabolite 1-methyl-4-phenylpyridine have melanin affinity, and the parkinsonism-inducing potency of MPTP is much stronger in species with melanin in the nerve cells. Autoradiography of [3H]MPTP in experimental animals has shown accumulation in melanin-containing tissues, including pigmented neurons. In the present whole body autoradiographic study accumulation and retention was seen in neuromelanin in frogs after i.p. injection of [14C]paraquat or [14C]diquat. By means of whole body autoradiography of [14C]diquat in mice (a species with no or very limited amounts of neuromelanin) a low, relatively uniformly distributed level of radioactivity was observed in brain tissue. Accumulation of toxic chemical compounds, such as paraquat, in neuromelanin may ultimately cause lesions in the pigmented nerve cells, leading to Parkinsons disease.

Whole-Body Autoradiography

Ever since its introduction in the early 1950s whole-body autoradiography has been used quite extensively for mapping the fate of various substances in the body, especially in the fields of pharmacology and toxicology. A detailed description of the whole-body autoradiographic technique is given. Some factors of general interest are dealt with—such as radionuclides and labeled substances, followed by a number of illustrations providing details and specific references.

Studies on the binding of chlorpromazine and chloroquine to melanin in vivo

Abstract Recent experiments have shown that the complex-formation of chlorpromazine and chloroquine with melanin in vitro comprises an electrostatic binding of the positively charged drug molecules to negative groups of the melanin-polymer (probably carboxyl-groups), supplemented by additional forces, which may involve van der Waals binding or charge-transfer complexes. The aim of the present study was to determine if a similar binding-mechanism may be present in vivo . Pigmented mice were injected with [ 35 S]chlorpromazine or [ 14 C]chloroquine. At 1 hr and 1 day the eyes were excised and the ability of cationic solutions to release the radioactivity from the pigment of eye-tissue preparations was determined by liquid scintillation counting. It was found that the cations were able to release the radioactivity from the pigment, with increasing effectiveness at increasing concentrations. The order of the ability of the cations to release the radioactivity was H + > Ca 2+ > Na + . A part of the radioactivity (for [ 35 S]chlorpromazine about 21 per cent, for [ 14 C]chloroquine about 8 per cent) was non-extractable. Similar results were obtained at the 1 hr and 1 day survival intervals. These results indicate that the binding-mechanism described above for the in vitro affinity is valid also for the situation in vivo .

Binding of paraquat and diquat on melanin

The binding on melanin of the quaternary dipyridylium salts paraquat and diquat has been studied in vivo in mice by autoradiography and in vitro by reaction with pigment from beef eyes. It was shown that paraquat and diquat were strongly bound on melanin in vivo and in vitro. The in vitro experiments showed that the uptake was concentration dependent. Experiments in vitro with paraquat demonstrated that the uptake was inhibited by Na+ and K+. Divalent cations also inhibited the binding of paraquat on melanin, the inhibiting ability being greater than for the monovalent cations. H+ effectively inhibited the binding. The results indicate that ionic binding is involved in the uptake of these compounds on melanin and this in turn can be ascribed to a cation-exchange activity of the melanin. Further studies are required to establish whether this may be a more general mechanism for the accumulation of drugs and chemicals on melanin.

The binding of inorganic and organic cations and H+ to cartilage in vitro

Abstract The binding of H + , Na + , Ca 2+ , Ni 2+ , Ce 3+ , chlorpromazine, chloroquine, hexamethonium, paraquat and gentamicin to decalcified cartilage from bovine nasal septum has been studied in vitro . The results indicated that the chondroitin sulphate is the binding material in the cartilage and that a stoichiometric binding occurs to the carboxyl and ester sulphate groups of the chondroitin sulphate. An analysis of the binding by the method of Scatchard was performed. The H + ions were bound to two groups of sites, one representing the carboxyl, the other representing the ester sulphate groups. It was shown that the Scatchard association constants for H + may be converted to p K a values. Such transformation showed that the carboxyl groups of the chondroitin sulphate had a p K a value of 4.57 and the ester sulphate groups a p K a value of 2.60. The data for Na + indicated a binding to three groups of sites. Two of these may represent the carboxyl and ester sulphate groups. The third, which was a small group with a very strong affinity, may represent a specific localised link with a few groups on the chondroitin sulphate. The experiments with Ca 2+ , Ni 2+ , Ce 3+ and the organic substances indicated that, in contrast to H + and Na + , only one binding class was present, which implies that the binding to the carboxyl and ester sulphate groups for these ligands occurs with a similar strength. The affinity of these cations to the cartilage was related to their positive charge, which is the general characteristic for an electrostatic interaction.

Present status of boron neutron capture therapy

The neutron capture reaction 10B(1n,4He)7Li produces two energetic particles, 4He2+ and 7Li3+ that are strongly cell toxic. Due to the short range of these nuclear fragments (5-9 microns) mainly those cells that have bound or internalized a 10B-containing substance are growth-inactivated. The most critical and difficult step in an efficient boron neutron capture therapy (BNCT) is the tumour targeting. It is today possible to synthesize a large number of boron compounds and conjugate them to tumour-seeking macromolecules, such as monoclonal antibodies or different polypeptides. The boron-containing substances presently considered for therapy are sulfhydryl boron hydride (BSH) and boron-phenylalanine, (BPA) for the treatment of gliomas and malignant melanomas respectively. Other boronated compounds considered are ligands for receptor-amplified tumour cells, antibodies for tumour cells with specific antigens and thioureas for treatment of melanotic melanomas. The required boron concentration is given by the relative dose due to neutron capture in 10B and that of the competing capture reactions in nitrogen and hydrogen. Capture in nitrogen produces protons with a range of about 10-11 microns and this gives a radiation dose to all cells in the neutron activated area. Calculations show that the local concentration of 10B near the critical radiation target, DNA, must be higher than 10 ppm (10 micrograms/g). Increased emphasis will be put on the development of combinations of treatments that fulfil the requirements for attacking the microscopic spread of the tumour.

COMPUTER-ASSISTED QUANTIFICATION AND IMAGE PROCESSING OF WHOLE-BODY AUTORADIOGRAMS

A computerized image-processing system especially adapted for analysis of whole-body autoradiograms has been developed. It consists of commercially available standard components, including a black-and-white video camera, a microcomputer, and graphics equipment. The lower performance of the hardware has been compensated for by more flexible software. When the system was calibrated, special attention was paid to local variations in the measuring system in different parts of the picture. Utility programs for the manipulation of contrast, pseudocoloring, and image enhancement, etc., are available. Some programs have been especially designed to comply with specific problems and demands related to different autoradiographic applications. A program displaying the density histogram for an area of interest is particularly useful for the quantitation of whole-body autoradiograms. It allows the operator to select interactively a range of densities. Image elements (pixels) corresponding to the densities in this range are shown in red on the monitor, and their average true density is calculated. This procedure permits the marking and analysis of delicate structures on autoradiograms. Other programs allow a picture, stored in memory, to be rotated or translated, and two pictures to be superimposed for comparison. Various applications of using image analyses in whole-body autoradiography are presented and illustrated.

Low Density Lipoprotein as a Carrier of Cytostatics in Cancer Chemotherapy: Study of Stability of Drug-carrier Complexes in Blood

Abstract 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.

On the binding of the bisquaternary ammonium compound paraquat to melanin and cartilage in vivo

Abstract The bisquaternary ammonium compound paraquat has been shown to accumulate in melanin-containing tissues and in cartilage in vivo . The present study was intended to elucidate if ionic binding is involved in the mechanism for the affinity of paraquat to these tissues in vivo . Ionic binding has previously been shown to be involved in the binding of paraquat to melanin in vitro and in the binding of other bisquaternary ammonium compounds to cartilage in vitro . [ 14 C]paraquat was given to mice. Whole-body tissue sections were taken and incubated in solutions with different cationic composition. The ability of the cations to displace [ 14 C]paraquat from melanin of the eye and intervertebral cartilages was then determined by autoradiography, using densitometric measurements for the quantitations. It was found that the cations in the incubation solutions were able to displace [ 14 C]paraquat from both melanin and cartilage. The [ 14 C]paraquat was more effectively displaced from cartilage than from melanin. In both tissues divalent cations were more effective than K + and Na + ; and H + was also effective. The results indicate that ionic binding is involved in the binding in vivo of paraquat both to melanin and cartilage. The binding sites may be carboxyl groups present in the subunits in melanin and ester sulphate and carboxyl groups of chondroitin sulphate in cartilage. The stronger binding of paraquat to melanin may depend on extra interactions due to conjunctions of the aromatic rings of the paraquat and the melanin polymer.