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.

Summary The in vivo characteristics of four analogues of benzoporphyrin derivative (BPD) (Brasseur et al., 1988; KreimerBirnbaum, 1989; Morgan et al., 1987a,b). Undoubtedly, the knowledge of these characteristics could help in selecting and/or designing photosensitisers for PDT. It appears likely, however, that no photosensitiser will have all of the desirable properties since no doubt, efficacious PDT is dependent on a large number of variables.We have been working with a chlorin-like photosensitiser, benzoporphyrin derivative (BPD), which is composed of four structural analogues following synthesis. All four analogues have an identical reduced tetrapyrrol porphyrin ring. They differ in two regards; the position of a cyclohexadiene ring which is fused at either ring A or B of the porphyrin, or in the presence of two acidic groups, or one acid and one ester group located at positions C and D of the porphyrins. In vitro characteristics of these analogues have been reported earlier (Richter et al., 1990a). Monoacid analogues were found to be more efficient photosensitisers than the diacids. Of the two monoacids, monoacid ring B (BPD-MB) was found to be slightly more soluble than the ring A analogue (BPD-MA) and preliminary in vivo work indicated that this could be a problem in obtaining reliable data because of the possible presence of aggregates in formulated materials. Therefore our studies on in vivo photosensitisation with BPD have focused largely on BPD-MA. The results are reported in this paper. We also report the results of limited studies carried out with other BPD analogues, undertaken in order to identify special characteristics which make a molecule of a photosensitiser efficient in in vivo PDT. Materials and methodsSynthesis of BPD analogues BPD was synthesised as described earlier . The length of hydrolysis of the dimethylester of A-ring or B-ring isomers with 25% hydrochloric acid dictates the final ratio between mono and diacids formed, longer hydrolysis leading to more complete conversion to the diacid form. The separation of the diacids from the monoacid analogues was carried out using column chromatography on silica gel as described earlier (Richter et al., 1990b). The following analogues were obtained: BPD-monoacid, ring A (BPD-MA), and ring B (BPD-MB), and BPD diacid, ring A (BPD-DA) and ring B (BPD-DB).All four BPD analogues were maintained in dimethyl sulfoxide (DMSO) at a concentration of 8 mg ml-'. Immediately before injection into the animals they were diluted in phosphate buffered saline (PBS). The injected solution contained no more than 10% DMSO.Tritiated BPD analogues Batches of BPD-MA, -MB and -DA were tritiated by NEN (Boston, Mass.) according to the procedure described earlier (Richter et al., 1990b). Two batches of BPD-MA were labelled and the specific activities were 5.9 mCi mg-' (1st batch) and 5.46 mCi mg-' (2nd batch). Specific activities of BPD-MB and BPD-DA were 9.2 mCi mg-' and 6.57 mCi mg-', respectively. The labelled compounds were tested for purity and photosensitising...

Congenitally thymusless nude mice that lacked functional T cells were reconstituted with H-2-compatible or -incompatible thymus grafts taken from either fetal, newborn, or adult mice and transplanted under the kidney capsule or subcutaneously. Transplantation with unirradiated fetal (15--17 d) or newborn thymus grafts reconstituted the nude mice as assessed by their subsequent generation of virus-specific cytotoxic T cells in vivo or alloreactive T cells in vitro. The restriction specificity of T cells from homozygous mice was exclusively for the nude host H-2, as shown by direct cytolysis or by cold target competitive inhibition assays. irrespective of whether nude mice were reconstituted with H-2-compatible, semiallogeneic, or H-2-incompatible, unirradiated newborn or fetal thymus grafts (in order of decreasing efficiency of reconstitution). The restriction specificity for the nonhost H-2 of the thymus could not be demonstrated even after primary or secondary sensitization in an infected appropriate F1 environment. These nude mice reconstituted with fetal or newborn grafts were tolerant to the H-2 of the thymus donors. Nude mice transplanted with irradiated adult thymus grafts were reconstituted functionally with syngeneic or semisyngeneic but not with allogeneic thymus grafts. In homozygous nu/nu irradiated heterozygous recipients of F1 thymus grafts, the restriction specificity for the nonhost thymic H-2 could not be elicited upon adoptive sensitization in irradiated and infected F1 heterozygote stimulator mice; in fact, these chimeras' lymphocytes were not tolerant to the nonhost H-2. The discrepancy between the restorative capacity of unirradiated vs. irradiated thymus grafts suggests that precursors of T cells in nude mice can acquire restriction specificity and immunocompetence independently of a conventional, functioning H-2-compatible thymus if exposed to an allogeneic fetal or a newborn thymus that contains functioning thymocytes of donor type but not if reconstituted with an irradiated adult allogeneic thymus.

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 M1 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 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 vivo/in vitro cytotoxicity assay performed 3 h post intravenous drug administration. Eight hours following photosensitizer injection only low density lipoprotein (LDL) mixtures produced significant (P less than or equal to 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 vivo/in 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.