Photodynamic therapy comes of age.

The concept underlying photodynamic therapy (PDT) is the use of light-absorbing molecules which, when delivered to target cells, and activated by irradiation with light of the appropriate wavelength, produce reactive oxygen species that cause cell death by apoptosis or necrosis. Classically, photodynamic agents have been macrocycles and, as such, application is limited to topical and intravenous administration. In the latter case, reliance has been placed on the characteristic behavior of the photodynamic agents in showing some degree of selectivity for concentrating in the target to minimize non-specific damage to the host tissue. The parameters open to development of improved drugs are: (i) the design of the photodynamic agent molecule as a means of determining the wavelength of light for activation, and for influencing physicochemical characteristics and the pharmacokinetic behavior of the drug; (ii) the delay between administration and activation; (iii) the nature of the activating light source; and, (iv) the duration and intensity of the activating light. Obviously, PDT is attractive for treating disease states in which natural apoptotic mechanisms are compromised, specifically for cancerous states and in cases of uncontrolled cell proliferation. PDT also has immunomodulatory sequelae, including triggering of T-cell mediated activity against residual cancerous cells. The use of PDT is being extended to diverse, related immune and proliferative disease states, and to the inactivation of bacteria and viruses. Increasingly, attention is being given to improving treatment by targeting conjugates and local delivery strategies, as well as by the design of photodynamic agents with closely defined photophysical and physicochemical properties. Progress is being made in challenging indications, such as the treatment of solid and pigmented tumors. Alternative technologies not involving light activation are available for some molecules which may also be used with light activation. Some ex vivo techniques and medical devices have been reported.

[3H]CGP 39653: a new N-methyl-D-aspartate antagonist radioligand with low nanomolar affinity in rat brain.

CGP 39653 (D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid) was initially discovered to inhibit the binding of [3H]L-glutamate and [3H]3-[+/-)2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid [( 3H]CPP) with Ki values of 230 and 5 nM, respectively. The radiolabeled compound [3H]CGP 39653 binds to rat frontal cortical membranes in a saturable and reversible manner. Analysis of saturation experiments revealed that the ligand labels one binding site with a Kd value of 6 nM. Competition experiments indicated that the order of potency of a number of competitive excitatory amino acid agonist and antagonist compounds was similar to that found previously for other N-methyl-D-aspartate (NMDA) receptor ligands. In contrast to these competitive inhibitors, which produced steep inhibition curves, glycine inhibited binding in a complex manner. When the functional activity of the unlabeled compound was explored, CGP 39653 blocked NMDA-evoked depolarizations in the rat cortical wedge in vitro and inhibited L-glutamate stimulated [3H]N(1-[2-thienyl]cyclohexyl)3,4-piperidine [( 3H]TCP) binding in cortical membranes. These results suggest that [3H]CGP 39653 selectively binds to the NMDA receptor as an antagonist with high affinity and is currently the ligand of choice for labeling the NMDA receptor.

Specific labeling of rat brain substance P receptor with [3H]physalaemin.

The binding of [3H]physalaemin [( 3H]PHY) to rat brain membranes is specific, saturable and reversible in the presence of monovalent cations and peptidase inhibitors. Monovalent cations increase the binding of [3H]PHY in an ionic strength (mu)-dependent manner with an optimal effect at mu higher than 0.3. Addition of 2.5 mM MnCl2 results in a 2-fold increase in the affinity (KD) and a 40% increase in the maximal receptor density (Bmax). Scatchard analysis under these conditions indicates the existence of a single population of noninteracting sites with KD of 3.6 nM and a Bmax of 76 fmol/mg of protein. Substance P (SP) and physalaemin are equipotent in inhibiting the binding of [3H]PHY, whereas the potency of SP(2-11), SP(3-11), and SP(4-11) decreased in inverse proportion to their length. The relative affinity of the different tachykinins, SP, and SP fragments in competing with [3H]PHY correlates with their potency to stimulate several bioassay systems, indicating that [3H]PHY labels a physiologically relevant binding site that correspond to the SP-P tachykinin receptor. Guanine nucleotides completely abolish the increase in the binding of [3H]PHY produced by 2.5 mM MnCl2, but in its absence, the nucleotides reduce binding only by 15%. Guanine nucleotides reduce binding to the same level regardless of the presence or absence of the divalent cation. Regional distribution studies confirm that the density of SP receptors is maximal in the olfactory bulb, followed by the hypothalamus, striatum, hippocampus, cortex, and cerebellum.

Distribution and fate of free and liposome-encapsulated [3H]nor-muramyl dipeptide and [3H]muramyl tripeptide phosphatidylethanolamine in mice.

The pharmacokinetics and metabolism of i.v. administered free (unencapsulated) or liposome-encapsulated hydrophilic [3H]-labeled nor-muramyl dipeptide (nor-MDP) and lipophilic [3H]-labeled muramyl tripeptide phosphatidylethanolamine (MTP-PE) were evaluated. In addition we also examined the distribution and fate of these immunomodulators subsequent to intranasal (i.n.) administration. Unique patterns of circulatory clearance, organ distribution, metabolism, and excretion were observed for each of the four preparations. Nor-MDP in saline was rapidly cleared from the circulation and excreted in the urine as intact molecules. MTP-PE dissolved in saline was cleared from the circulation at a slow rate and found within various organs as intact MTP-PE, lyso-MTP-PE, and MDP. Following the i.v. administration of nor-MDP or MTP-PE in liposomes, patterns of clearance and organ distribution corresponded to that of liposome distribution, i.e., the reticuloendothelial system. Extensive dissociation of hydrophilic nor-MDP from the carrier liposomes occurred, and the immunomodulator was recovered in the urine. In contrast, MTP-PE entrapped in liposomes was retained in target organs for the duration of the study. The i.n. instillation of radiolabeled nor-MDP or MTP-PE was associated with the accumulation of these immunomodulators in the brain. Our results demonstrate the feasibility of targeting hydrophilic and lipophilic immunomodulators to cells of the macrophage-histiocyte series.

Rat brain membranes possess two high-affinity binding sites for [3H]somatostatin.

This report described the first use of [4-3H-Phe6]somatostatin-14 to characterize binding sites on rat brain membranes for somatostatin-14. This ligand is superior to previously used iodinated analogues and is chemically and biologically identical to the natural ligand. Two high-affinity binding sites were found, from Scatchard analysis of competitive displacement experiments, with Kd SS1 = 0.41 and Kd SS2 = 22.9 nM. Specific binding was reversible, and kinetic analysis of the dissociation and association time-course gave an apparent Kd of 0.44 nM, in good agreement with the Kd of the higher-affinity site. Specific binding of the ligand was enriched in cerebral cortex and hippocampus, with intermediate levels in the striatum, hypothalamus and midbrain, and low levels in the pons/medulla and cerebellum. This ligand should prove to be valuable for elucidating the physiological and pharmacological significance of the two subtypes of somatostatin binding sites we have demonstrated.

Specific binding of [3H-Tyr8]physalaemin to rat submaxillary gland substance P receptor.

[3H]Physalaemin [( 3H]PHY) binds to a single class of noninteracting sites on rat submaxillary gland membranes suspended in high ionic strength media with a KD of 2.7 nM, a Bmax of 240 fmol/mg of protein, and low nonspecific binding. The relative potencies of substance P (SP) and its fragments in competing with [3H]PHY correlate with their relative salivation potencies. This indicates that [3H]PHY interacts with a physiologically relevant SP receptor. In low ionic strength media, the KD of [3H]PHY does not change, but SP and some of its fragments are more potent than PHY in competing with [3H] PHY. Computer-assisted analysis of [3H]PHY and [3H]SP binding in high and low ionic strength media demonstrated that both peptides are equipotent in high ionic strength but that the affinity of SP increases by 70-fold in low ionic strength. The SP fragments that contain a basic residue in positions 1 and/or 3 also display an increased affinity in low ionic strength. These findings document that [3H]PHY binding in high ionic strength (mu = 0.6) accurately reflects the pharmacological potencies of agonists on the SP-P receptor. The binding of [3H]PHY, like that of [3H]SP, increases by the addition of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+). Guanine nucleotides decrease [3H]PHY binding by decreasing the Bmax to the same level (160 fmol/mg of protein), in the presence or absence of Mg2+.

[7-Methyltryptophan9]-beta-corticotropin-(1-24): a hyperactive Synacthen analogue.

[7-Methyltryptophan9]-beta-corticotropin-(1-24) has been synthesised. In an isolated adrenal cell bioassay, it has 2.7 times the steroidogenic activity of beta-corticotropin-(1-24) (Synacthen).

Tritiated peptides. 12. Synthesis and biological activity of [4-3H-Phe8]substance P.

Substance P has been prepared 3H labeled at Phe8 by catalytic deiodination of a protected precursor. Synthesis of the precursor was by solid-phase methodology on polydimethylacrylamide resin and by condensation in solution of fragments covering sequences 1-4, 5-7, and 8-11. Free peptide made by each route analyzed satisfactorily and had the same chromatographic characteristics as unlabeled substance P. It was indistinguishable from the latter by radioimmunoassay when N and C terminally directed antisera were used and in the ability to cause contractions of isolated guinea pig ileum. Specific radioactivity was 23 Ci/mmol.

CGS 8216: receptor binding characteristics of a potent benzodiazepine antagonist.

CGS 8216 is a novel nonbenzodiazepine that inhibited 3H-flunitrazepam (3H-FLU) binding to rat synaptosomal membranes in vitro at subnanomolar concentrations. It prevented the in vivo labeling of brain benzodiazepine receptors by 3H-FLU with the same potency as diazepam when given orally to mice. Pharmacologic tests showed that it was devoid of benzodiazepine-like activity but it antagonized the actions of diazepam in these tests. It did not interact with alpha- or beta- adrenergic, H1-histaminergic or GABA receptors but it inhibited adenosine-activation of cyclic AMP formation. Studies with 3H-CGS 8216 demonstrated that it bound specifically and with high affinity to rat forebrain membranes and was displaced by drugs with an order of potencies similar to that observed when 3H-diazepam and 3H-FLU were used as radioligands. The regional distribution of 3H-CGS 8216 binding sites in the brain was also similar to that of 3H-FLU. Dissociation of 3H-CGS 8216 binding was slow at 0 degrees C but increased with temperature and was almost complete within 1 min at 37 degrees C. Scatchard analyses were linear, yielding KD values of 0.044, 0.11 and 0.18 nM at 0, 25 and 37 degrees C, respectively; the Bmax value did not change appreciably with temperature and was approximately 1000 fmoles/mg protein. Using 3H-FLU, the value for Bmax as well as for the KD increased with temperature. The total number of binding sites determined for 3H-FLU was greater than that for 3H-CGS 8216 at each temperature. CGS 8216 exhibited mixed-type inhibition of 3H-FLU binding. GABA did not stimulate 3H-CGS 8216 binding whereas it enhanced 3H-FLU binding. CGS 8216 may be a useful ligand for probing the antagonist properties of the benzodiazepine receptor and is likely to exhibit interesting therapeutic effects.

Specific binding of 3H-substance P to rat brain membranes.

The undecapeptide substance P is a putative neurotransmitter in the mammalian central nervous system (CNS), and may be associated with pain fibres in the spinal cord. Radiolabelled derivatives of other neuropeptides have been used to demonstrate specific interactions with receptor sites on brain membranes, and this approach has now been explored with substance P. We have now prepared [4-3H-Phe8]-substance P and we find that it binds reversibly to a saturable population of sites in rat brain particulate fractions. Scatchard analysis of concentration-dependent saturation of binding indicates a single population of non-interacting sites with a high affinity (Kd=0.38 nM) and a low density (Bmax=27.2 fmol per mg protein). Kinetic analyses indicate an apparent dissociation equilibrium constant of 0.46 nM. A variety of neurotransmitter amines and amino acids, and other peptides do not compete at the substance P sites, but structurally related peptides or shorter C-terminal fragments of substance P are active. The rank order of potency of these substance P-related peptides agrees with that reported for their effects in depolarizing spinal cord neurones. The regional distribution of the specific binding sites for 3H-substance P parallels that of substance P immunoreactivity, being high in the hypothalamus and low in the cerebellum and cerebral cortex. The characteristics of the 3H-substance P binding sites are consistent with those expected for substance P receptors.

The synthesis of tritium-labelled human corticotropin of high specific radioactivity.

Human [[3,5-3H2]Tyr23]corticotropin-(1-39)-nonatriacontapeptide of specific radioactivity 25.2 Ci/mmol, identical with unlabelled human corticotropin by several criteria, was prepared via the fully protected di-iodotyrosine compound. The latter was synthesized by classical procedures.

Pneumococcal C-substance, a ribitol teichoic acid containing choline phosphate.

1. Pneumococcal C-substance was isolated from the non-capsulated Pneumococcus 1-192R, A.T.C.C. 12213, by extraction with trichloroacetic acid solution followed by chromatography on DEAE-cellulose (HCO(3) (-) form). 2. The polymer contains 7.0% of phosphorus and 6.0% of nitrogen and is composed of phosphate, N-acetyl-d-galactosamine, d-glucose, N-acetyldiaminotrideoxyhexose, ribitol and choline in the molecular proportions 2:1:1:1:1:1. 3. After acid hydrolysis, d-galactosamine hydrochloride and galactosamine 6-phosphate were isolated in crystalline form and crystalline derivatives of d-glucose and anhydroribitol were obtained. A product of partial acid hydrolysis was provisionally characterized as 6'-O-phosphoryl-[O-beta-d-galactosaminyl-(1'-->6)-d-glucose]. 4. C-substance contains free amino groups accessible to attack by 1-fluoro-2,4-dinitrobenzene and nitrous acid. 5. Choline phosphate and ribitol phosphate are units in the polymer. 6. Treatment with hot alkali gave a fragment comprising phosphate, d-galactosamine, d-glucose, diaminotrideoxyhexose and ribitol in the molecular proportions 2:1:1:1:1. 7. After selective N-acetylation, the fragment contained one of its phosphate groups as a phosphomonoester and one as a phosphodiester, shown by potentiometric titration and by treatment with a phosphomonoesterase. 8. C-substance from seven other strains of Pneumococcus possesses a structure common to that described for the strain 1-192R. 9. Capsular materials from 26 different strains of Pneumococcus were analysed for suspected contamination by C-substance. In 19 cases the presence of C-substance with the normal structure was demonstrated, and in the remaining seven cases the contaminating C-substance was probably similarly constituted. 10. F-substance was isolated and the associated fatty acid material analysed.

The structure and possible function of the glycolipid from Staphylococcus lactis I3.

1. The total lipid was extracted from Staphylococcus lactis I3 with chloroform-methanol mixtures and the glycolipid component was isolated by chromatography on silicic acid. 2. Saponification yielded a non-crystalline glycoside for which the structure O-beta-d-glucopyranosyl-(1-->6)-O-beta-d-glucopyranosyl-(1-->1)-d-glycerol has been established by chemical degradations and by comparison with synthetic material. 3. The role of the glycosyl diglycerides in bacterial membranes is discussed.