Julia G. Levy

An immunocytochemical investigation with monoclonal antibodies to somatostatin

SummaryFour monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin.The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.

Liposomal delivery of a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD), to tumor tissue in a mouse tumor model.

Abstract— Biodistribution studies were carried out on 14C‐labeled benzoporphyrin derivative monoacid ring A (BPD), which had been formulated as a unilamellar liposome or taken from a stock solution in dimethyl sulfoxide diluted into phosphate‐buffered saline immediately before intravenous injection into DBA/2 mice. By and large the general distribution of BPD to various organs and tissues was comparable for both formulations. It was noted, however, that liposomal material appeared to enter tissues more rapidly and to be cleared more rapidly, as demonstrated by shorter half‐lives for a number of tissues including skin, lung and fat, and generally lower levels in most tissues 24 h following administration. Accumulation in tumor tissue was slightly higher with liposomal BPD, and clearance rates for this tissue were equivalent (half‐lives 16.1 h for liposomal BPD and 16.9 h for aqueous BPD). When the two preparations were tested in a bioassay in tumor‐bearing mice, photodynamic therapy (PDT) with liposomal BPD proved to be superior to the aqueous preparation when PDT was administered 3 h following intravenous administration of BPD. Plasma distribution studies in vitro demonstrated that 91.1 ± 0.3% of the liposomal BPD distributed to the lipoprotein fraction within the first hour of mixing, whereas only 49.1 ± 2.6% of nonliposomal BPD was associated with lipoprotein under the same conditions. Furthermore, while lipoprotein‐associated liposomal BPD distributed evenly between all three types of lipoprotein (high, low and very low density), a majority of nonliposomal BPD associated with the high‐density lipoprotein fraction.

Biodistribution of tritiated benzoporphyrin derivative (3H-BPD-MA), a new potent photosensitizer in normal and tumor-bearing mice

The biodistribution of a new and very potent photosensitizer, benzoporphyrin derivative-monoacid, ring A (BPD-MA), was determined in normal and P815 (mastocytoma) or M1 (rhabdomyosarcoma) tumor-bearing DBA/2J mice. A dose of 80 micrograms of 3H-BPD-MA was determined at 3, 24, 48, 72, 96 and 168 h post injection. The following tissues were tested: blood, brain, heart, intestine, kidney, lung, liver, muscle, skin, stomach, spleen, thymus and tumor. The biodistribution of 3H-BPD-MA in normal and tumor-bearing mice was comparable overall. 3H-BPD-MA localized in tumors better than in other tissues except kidney, liver and spleen. The tumor to tissue ratios were in the range 1.5-3 at 24 h post injection and increased further during the next 72 h. The highest levels of 3H-BPD-MA were observed in all tissues at 3 h post injection and decreased rapidly during the first 24 h. After 24 h the clearance from tissues was rather slow. The preliminary clearance data obtained in a group of five normal mice indicated that the majority of the injected dose (60%) cleared from the body via the bile and feces, while only about 4% cleared via kidneys and urine. Studies in which 3H-BPD-MA was extracted from tumor, kidney and liver 3 and 24 h after injection showed that, at 3 h, all the photosensitizing activity in tumor was retained. At 24 h only 39% of the activity was retained and considerably less active material was present in liver and kidney.

The effects of plasma lipoproteins on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative.

The influence of lipoprotein association on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative (BPD) has been investigated in M‐1 tumor bearing mice. The association of benzoporphyrin mono acid ring A with either low or high density lipoprotein increased tumor cell killing in an in vivolin vitro cytotoxicity assay performed 3 h post intravenous drug administration. Eight hours following photosensitizer injection only low density lipoprotein (LDL) mixtures produced significant (P≤ 0.005) increases in tumor cell killing compared to BPD in unfractionated plasma. The efficacy of in vivo photosensitization in the presence of lipoproteins correlated with the in vivolin vitro cytotoxicity. Association of BPD with low or high density lipoproteins resulted in delayed tumor regrowth and higher cure rates when light exposure (125J/cm2) was performed 3 h post drug administration. When light exposure was performed 8 h post‐injection only LDL‐BPD mixtures led to enhanced tumor eradication compared to BPD administered in aqueous solution or unfractionated plasma.

Photosensitizers as virucidal agents.

The photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) has been studied regarding its ability to destroy enveloped viruses in blood and blood products when activated by light. Its maximum wavelength of absorption (690 nm) has proven useful in terms of activation of the photosensitizer in materials containing red blood cells. Experiments conducted on whole blood of patients infected with the human immunodeficiency virus (HIV) demonstrated that BPD-MA and light could effectively eliminate the virus when treated materials were placed in culture and tested for viral p24, but did not appear to damage blood cells or blood components. Since HIV is largely intracellular in infected individuals, these results were investigated further. We have shown, using flow cytometry, that in HIV-infected blood, BPD-MA and light appear to selectively destroy white cells that bear the interleukin 2 receptor and the DR antigen. These markers are prevalent on activated lymphocytes, and since HIV replicates only in CD4+ T cells which are activated, this finding provides an explanation for the selective killing of HIV.

Photodynamic therapy induces caspase-3 activation in HL-60 cells.

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PKCS) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.

In vitro EVALUATION OF PHOTOTOXIC PROPERTIES OF FOUR STRUCTURALLY RELATED BENZOPORPHYRIN DERIVATIVES

Abstract— Four structural analogs of benzoporphyrin derivative (BPD) have been studied and compared for photosensitizing activity in vitro. All analogs have an identical reduced tetrapyrrol porphyrin ring, and differ by the position of a cyclohexadiene ring (fused at either ring A or ring B of the porphyrin) and the presence of either two acid groups or one acid and one ester group at rings C and D of the porphyrin. Photosensitizer activity was tested with the Ml tumor cell line using an assay (the MTT assay) which detects mitochondrial hydrogenases as a measure of cell viability. This assay was shown to be equivalent to the standard clonogenicity or [3H]thymidine uptake assay. Comparative studies with the BPD analogs showed that the monoacid derivatives had equivalent cytotoxicity and were about five‐fold more active than the diacid forms. This was the case whether the assays were performed in the presence or absence of fetal calf serum.

THE PLASMA DISTRIBUTION OF BENZOPORPHYRIN DERIVATIVE and THE EFFECTS OF PLASMA LIPOPROTEINS ON ITS BIODISTRIBUTION

Abstract— The plasma distribution and biodistribution of benzoporphyrin derivative were examined. Two analogs of benzoporphyrin derivative were mixed with human plasma in vitro and recovered in the lipoprotein fractions upon separation by chromatography or ultracentrifugation. The majority of both analogs was recovered with high density lipoprotein. The effect of prebinding benzoporphyrin derivative to lipoproteins on the biodistribution of the drug in vivo was studied in tumor bearing DBA/2J mice. At 3, 8 and 24 h post‐injection, tumor and tissue samples were excised and analyzed for benzoporphyrin derivative content. Precomplexing benzoporphyrin derivative with low density lipoprotein or high density lipoprotein led to significantly (P < 0.05) greater tumor accumulation than in aqueous solution.

New Applications in Photodynamic Therapy Introduction

Photodynamic therapy is now recognized as a legitimate therapy for both palliative and potentially curative treatment of solid tumors. This represents a major achievement for investigators who have committed their careers to PDT. Scientific research characteristically runs well ahead of accepted views and technologies. This Symposium-in-Print perhaps provides a snapshot of the potential for this technology during the next two decades.

Development of technology for linking photosensitizers to a model monoclonal antibody

A procedure is described whereby the photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) was covalently linked to a model monoclonal antibody in a manner which is reproducible, quantifiable, and retains both the biological activity of the antibody and the cytotoxicity of the photosensitizer. Preliminary steps involved the linkage of BPD-MA to a modified polyvinyl alcohol (PVA) backbone, followed by conjugation to the antibody using heterobifunctional linking technology. Briefly, polyvinyl alcohol (MW ca. 10,000) was modified with 2-fluoro-1-methyl pyridinium toluene-4-sulfonate and 1,6-hexanediamine to produce side chains containing free amino groups. The free carboxyl group of BPD-MA was utilized to conjugate photosensitizer molecules to modified PVA using a standard carbodiimide reaction. Final linkage of the PVA-BPD to a model monoclonal antibody involved further substitution of the carrier with 3-mercaptopropionic acid and carbodiimide to introduce 3-4 sulfhydryl residues per carrier molecule, and introduction of sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester residues to the monoclonal (3-4 residues/molecule). Conjugation was effected by reaction of the two species at pH 5.5 for 18 h. Detailed methodology and tests for efficacy of the procedure are provided.