Richard C. Straight

Geographical variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) venom properties

Individual venom samples were analyzed from 12 specimens of Crotalus scutulatus scutulatus, from north of Tucson to the extreme southeastern region of Arizona. Six of the specimens, from north of Tucson, produced venom lethal toxicity (i.p. LD50) values in mice of 2.0-6.0 mg/kg. These coincided with the values previously reported for C. s. scutulatus in the Phoenix, Arizona, region and designated as type B venom (Glenn and Straight, 1978). In contrast, the venom LD50 of six individuals from extreme southeastern Arizona, including one individual near Tucson, ranged from 0.22-0.46 mg/kg. This corresponds to the values for C. s. scutulatus venom previously reported and designated as type A venom (Glenn and Straight, 1978). Specimens with type A venom have been collected in California, Nevada, Utah and regions of Arizona. In addition to differences in lethal toxicity, the type B venom consistently exhibits a different protein profile, greater proteolytic activity, greater hemorrhagic activity and contains little or none of the major lethal toxin, Mojave toxin, compared to the type A venom. No external morphological characteristic could be found differentiating the type A venom specimens from the type B venom specimens. These findings further confirm the geographical variation of C. s. scutulatus venom in Arizona.

A polymeric drug delivery system for the simultaneous delivery of drugs activatable by enzymes and/or light

Three water soluble copolymers based on N-(2-hydroxypropyl)methacrylamide were prepared. Copolymer I contains adriamycin, a chemotherapeutic agent, attached via enzymatically degradable oligopeptide (glycylphenylalanylleucylglycine; G-F-L-G) side chains. The other two copolymers contained the photosensitizer, meso-chlorin e6 monoethylene diamine disodium salt (Mce6). In Copolymer II, the chlorin is attached via the degradable G-F-L-G sequence, and it was bound by the nondegradable glycyl spacer in Copolymer III. Initially, the copolymers were characterized separately in vitro and in vivo. Combinations of the copolymer bound chemotherapeutic agent and each of the copolymer bound photosensitizers were then assessed for antitumor effect in vivo. Localization/retention studies (A/J mice; Neuro 2A neuroblastoma solid tumor) were performed with the two copolymers containing Mce6 as well as the free drug. Results of these experiments demonstrated a very different tumor uptake profile for the two copolymers. While the free drug was rapidly cleared from tumor tissue, the copolymer containing Mce6 attached via the non-degradable bond was retained for an extended period; drug concentrations in the tumor were high even after 5 days. On the other hand, a high concentration of the copolymer containing Mce6 bound via the degradable sequence was taken up by the tumor, yet its concentration in the tumor was substantially diminished at 48 h after administration. This shows indirect evidence of in vivo cleavage of Mce6 from the copolymer in the lysosomal compartment which is supported by direct evidence of cleavage by cathepsin B (a lysosomal enzyme) in vitro. Antitumor effects were assessed on Neuro 2A neuroblastoma induced in A/J mice for all three copolymers. Photodynamic therapy (PDT) proved the copolymer with Mce6 bound via the degradable oligopeptide sequence to be a more effective photosensitizer in vivo than the other chlorin containing copolymer. The difference in activity was consistent with the results obtained by photophysical analyses in which the free drug had a higher quantum yield of singlet oxygen generation than the polymer bound drug in buffer. The quantum yield of singlet oxygen generation increased with the enzymatic cleavage of the chlorin from the copolymer. Conditions were subsequently determined for which chemotherapy or PDT would show some antitumor effect, yet be incapable of curing tumors. Finally, combination therapy experiments were performed in which the copolymer bound adriamycin was mixed with either of the copolymer bound chlorin compounds and injected intravenously (i.v.) into the tail veins of mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Combination chemotherapy and photodynamic therapy of targetable N-(2-hydroxypropyl)methacrylamide copolymer–doxorubicin/mesochlorin e6-OV-TL 16 antibody immunoconjugates

The aim of this study was to evaluate the combination chemotherapy and photodynamic therapy of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin (DOX) and mesochlorin e(6) (Mce(6)) targeted with an OV-TL 16 monoclonal antibody (P-DOX-Ab and P-Mce(6)-Ab, respectively) in nude mice bearing human ovarian OVCAR-3 carcinoma xenografts. P-DOX-Ab and P-Mce(6)-Ab were synthesized by first conjugating DOX or Mce(6) to an HPMA copolymer precursor (Mw=21000), then reacting with OV-TL 16 antibody. The immunoconjugates were purified by size exclusion chromatography on Superose 6 column and analyzed. The Mce(6) concentration in tissues was determined by a fluorescence assay. Eighteen hours after administration, the tumors received a light dose of 220 J/cm(2) from a KTP 650-nm dye-laser. P-DOX-Ab and P-Mce(6)-Ab had polymer:drug:protein weight ratios of 32:3:62 and 26:2:72, corresponding to polymer:drug:protein molecular ratios of approximately 4:14:1 and 3:8:1, respectively. The biodistribution results indicated that the percentage of total administered dose of Mce(6) in tumors reached approximately 1% for the nontargeted conjugate at 18 h after administration, while that of P-Mce(6)-Ab was approximately 13 times higher. Nude mice bearing OVCAR-3 xenografts that received one i.v. dose of P-DOX-Ab (2.2 mg/kg DOX equivalent) and P-Mce(6)-Ab (1.5 mg/kg Mce(6) equivalent) with light irradiation achieved a xenograft cure rate of more than 60%. The incorporation of OV-TL 16 antibody dramatically enhanced the accumulation in tumors with a concomitant increase in the therapeutic efficacy of P-DOX-Ab and P-Mce(6)-Ab in combination therapy, which may probably be attributed to both antibody targeting and enhanced permeability and retention (EPR) effects.

Systemic immunosuppression induced by photodynamic therapy (PDT) is adoptively transferred by macrophages.

Abstract— Some derivatives of hematoporphyrins are strongly retained by tumor tissue as compared to normal tissue, and exposure of these photosensitizers to radiation in the visible spectrum can cause serious biological damage. These properties have been exploited in the development of a new treatment for cancer termed photodynamic therapy (PDT). However, recent studies have also demonstrated that PDT can also induce a state of systemic immunosuppression. The purpose of this study was to determine whether PDT‐induced suppression of contact hypersensitivity (CHS) responses was an active phenomenon that could be adoptively transferred by viable splenocytes from PDT‐treated mice. Although induction of adoptively transferable suppressor cells in PDT‐treated mice required exposure to antigen, the suppressor cells were found to be antigen nonspecific in their function. Furthermore, splenocytes from PDT‐treated mice were capable of generating levels of allospecific cytotoxic T lymphocyte (CTL) activity which were comparable to those generated by normal control mice, but the ability of irradiated spleen cells from PDT‐treated mice to stimulate a mixed lymphocyte response (MLR) was dramatically impaired. Finally, chromatographic separation of T cells, B cells and macrophages showed that the cell type which mediates adoptively transferable suppression of CHS responsiveness is in the macrophage lineage.

Detection of myotoxin a-like proteins in various snake venoms

Ninety-five venom samples from eight snake genera (Agkistrodon, Bitis, Bothrops, Calloselasma, Crotalus, Sistrurus, Naja and Vipera) including venoms of Crotalus species of different geographical origin were assayed using immunodiffusion or an ELISA for the presence of the small basic protein, myotoxin alpha, known to cause muscle necrosis. Of the eight genera investigated, only Crotalus and Sistrurus venoms contained detectable amounts of myotoxin alpha-like proteins. The venoms of 13 out of 17 rattlesnake species investigated contained proteins immunologically similar to myotoxin alpha, including 12 Crotalus species and one Sistrurus species. The highest amounts were detected in venoms of C. exsul, C. viridis oreganus and C. v. viridis. Qualitative differences in the presence or absence of myotoxin alpha-like proteins were observed in the venoms of C. cerastes, C. horridus, C. lepidus, C. mitchelli, C. scutulatus, C. viridis and S. catenatus specimens of different geographic origin. The toxin was not detected in the venoms obtained from C. adamanteus, C. atrox, C. enyo or C. vegrandis specimens. The toxin appears to be widely distributed among rattlesnake species in the new world, but may vary qualitatively by geographical region in several species and subspecies.

Coherent backscattering in biological media: measurement and estimation of optical properties

We measured the coherent backscattering of light from milk solutions and biological tissues by using a He-Ne laser (633 nm) and a CCD array as a detector. A coherent peak from the milk solutions could bemeasured with a single exposure. However, ensemble averaging was required for coherent peaks to be produced from solid media such as tissue samples. By fitting experimental data to an existing model numerically, effective scattering and absorption coefficients were estimated. They were compared with those computed from integrating sphere measurements. Effective scattering coefficients computed by the two different methods were in good agreement for high-scattering media. However, higher absorption was estimated by the coherent peak method.

SENSITIZED PHOTOOXIDATION OF AMINO ACIDS: EFFECTS ON THE REACTIVITY OF THEIR PRIMARY AMINE GROUPS WITH FLUORESCAMINE AND O‐PHTHALALDEHYDE

Abstract— The photooxidation of his, met, trp and tyr with methylene blue and eosin as sensitizers resulted in a loss of primary amine reactivity as measured with fluorescamine and o‐phthalaldehyde. The rates of oxygen uptake and of loss of primary amine reactivity with illumination was characteristic for each amino acid. Glycine did not photooxidize and showed no change in primary amine reactivity. Primary amine reactivity of the N‐acetyl derivatives of these amino acids was negligible and during photooxidation the reactivity increased slowly with N‐acetylhis and N‐acetyltrp while the reactivity of N‐acetylmet and N‐acetyltyr did not change. Imidazole and indole also showed a slow increase in primary amine reactivity during sensitized photooxidation. The rate of oxygen uptake and primary amine reactivity loss during the sensitized photooxidation of his and met was markedly increased in deuterium oxide. This indicated that singlet oxygen was involved. Radical scavengers did not affect the rates indicating that free radical intermediates were not involved. Primary amine reactivity loss is probably due to intra‐ and/or inter‐molecular secondary dark reactions between unknown initial oxygenation products and primary amino groups.

Systemic immunosuppression induced by peritoneal photodynamic therapy

Although photodynamic therapy is being used increasingly for the diagnosis and treatment of human cancer, its effect on immune responses has received little attention. This aspect was examined in a murine model. Mice given peritoneal photodynamic therapy had markedly decreased contact hypersensitivity responsiveness, not observed with cutaneous photodynamic therapy. The immunosuppression was systemic, because contact hypersensitivity was depressed at distal, unirradiated sites. Photodynamic therapy induced adoptively transferable cells that inhibited contact hypersensitivity responses in naive mice. The immunosuppression was reversible, but persisted for 3 weeks after photodynamic therapy. An acute-phase response characterized by leukocytosis and elevated serum amyloid P levels was observed in mice given photodynamic therapy but not in mice treated with either laser or dye alone. These data suggest a link between the acute-phase response and immunosuppression. Thus, although photodynamic therapy shows promise in cancer treatment, the induction of decreased systemic immunoresponsiveness is an important observation with potentially detrimental consequences.

The midget faded rattlesnake (Crotalus Viridis Concolor) venom: Lethal toxicity and individual variability☆

Abstract The midget faded rattlesnake ( Crotalus viridis concolor ) venom: lethal toxicity and individual variability. Toxicon 15, 129–133, 1977.— Crotalus viridis concolor venoms were collected from captive specimens from northeastern Utah and tested for lethal potency in mice and for protein content by polyacrylamide gel electrophoresis. We found C. v. concolor potency (i.p.- ld 50 -0·25 μg per g) comparable to Crotalus durisses terrificus (i.p.- ld 50 -0·25 μg) and Crotalus scutulatus scutulatus (California variety) (i.p.- ld 50 -0·24 μg per g) venoms. Individual C. v. concolor venoms show a wide variability both in lethality and protein pattern. The venom appears to be one of the most lethal Crotalid venoms in the New World.

INACTIVATION AND MUTAGENESIS OF Herpes VIRUS BY PHOTODYNAMIC TREATMENT WITH THERAPEUTIC DYES

Dyes which photosensitize membranes may be clinically useful for photodynamic treatment (PDT) of Herpes simplex virus (HSV) infections. It is important to determine whether the enveloped HSV can be inactivated via membrane damage without affecting the genetic material. Selection of appropriate PDT conditions, including the choice of dye, could minimize viral mutagenesis. We determined the mutagenesis caused by PDT employing three membrane‐photosensitizing dyes of potential use in cancer photochemotherapy (Photofrin II, polyhematoporphyrin esters, zinc phthalocy‐anine tetrasulfonates) and a DNA‐photosensitizing dye (proflavine sulfate). The effects were compared to those caused by exposure of HSV to ultraviolet radiation(UV). The procedure consisted of incubating HSV with µ,g/ml (µM) concentrations of the dye, irradiating the samples with broad spectrum visible/near‐UV radition (Daylight fluorescent lamps) and assaying the survival of the treated HSV. Zinc phthalocyanine was the most potent dye per absorbed photon for inactivating HSV. In parallel with determination of survival, progeny of the surviving virus were grown for determination of mutagenesis. The progeny virus was harvested and subsequently assayed in the presence and absence of 40 µ,g/ml iododeoxycytidine (ICrd) to determine the frequency of mutation to ICrd resistance. Mutation frequencies were determined for progeny from the 1–4% survival level. For PDT with each membrane‐photosensitizing dye, only zinc phthalocyanine increased the mutation frequency over the untreated control. This increase was <2‐fold. Proflavine increased the mutation frequency 2–3 fold over the untreated control. Ultraviolet produced a 15–20 fold increase over the untreated control. Therefore, all four dyes were much less mutagenic than UV at the same toxicity level (P<0.0001), and the membrane‐photosensitizing dyes were less mutagenic than the DNA‐photosensitizing proflavine at the same toxicity level (P<.0001).