Neotropical Rattlesnake (Crotalus simus) Venom Pharmacokinetics in Lymph and Blood Using an Ovine Model.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

Phase I and pharmacokinetics study of crotoxin (cytotoxic PLA(2), NSC-624244) in patients with advanced cancer.

A Phase I clinical trial was performed on patients with solid tumors refractory to conventional therapy. Crotoxin was administered i.m. for 30 consecutive days at doses ranging from 0.03 to 0.22 mg/m(2). Patients entered the study after providing a written informed consent. Although 26 patients were entered only 23 were evaluated. Reversible, nonlimiting neuromuscular toxicity evidenced as diplopia because of pareses of the external ocular muscles was present in 13 patients. It started at doses of 0.18 mg/m(2) and lasted from 2 to 6 h. These episodes did not require dose adjustment and disappeared in 1-3 weeks of treatment. Three patients experienced palpebral ptosis, nystagmus (grade 2), and anxiety (grade 2-3) at the dose-limiting toxicity of 0.22 mg/m(2). Also at dose-limiting toxicity, 1 patient showed nystagmus (grade 2) and anxiety (grade 3) without evidence of palpebral ptosis. Transient increases (grades 1-3) in the levels of creatinine kinase, aspartate aminotransferase, and alanine transaminase attributed to crotoxin myotoxicity were observed but returned to normal by the last week of treatment. At 0.21 mg/m(2) there was a case of grade-3 anaphylactic reaction on day 31, which required treatment. Hypersensitivity was regarded as an adverse drug-related reaction, and the patient was removed from the protocol. Two patients at different doses (0.12 mg/m(2) and 0.22 mg/m(2)) had sialorrhea. Four patients had asymptomatic transient increase in blood pressure (up to 20 mm Hg) 12 h after the first injection, which lasted 24 h. No treatment was required and toxicity did not reappear. Six patients experienced slight eosinophilia during the first 2 weeks. The maximum tolerated dose was set at 0.21 mg/m(2). Objective measurable partial responses (>50% reduction of tumor mass) were noted in 2 patients treated at 0.21 mg/m(2) and 1 at 0.12 mg/m(2). One patient (at 0.21 mg/m(2)) presented a complete response on day 110. Crotoxin pharmacokinetics showed rapid absorption from the injection site to blood (t(1/2 A) = 5.2 +/- 0.6 min). Plasma concentration reached a peak (C(max) = 0.79 +/- 0.1 ng/ml) at tau(max) = 19 +/- 3 min. The half-life of the distribution (alpha) phase is 22 +/- 2 min. Starting at 1.5 h after injection, the decrease in plasma concentration becomes slower, reaching 14 +/- 3 pg/ml 24 h after injection. The profile is dominated by the elimination (beta) phase with a half-life of 5.2 +/- 0.6 h. Consequently, 24 h after the injection ( approximately 5 half-life) 97% of the product was eliminated. The area under plasma concentration versus time curve was 0.19 +/- 0.05 microg/min/ml. Assuming availability (F) approximately 1, the clearance is C(L) = 26.3 +/- 7 ml/min, and the apparent volume of distribution is V(d) = 12 +/- 3 liter/kg. The recommended dose for a Phase II study is 0.18 mg/m(2).

Differential biodistribution of native and 2 kGy 60Co irradiated crotoxin in tissues of CBA/J mice.

Crotalus durissus envenomation is treated using antivenins produced in horses. During production, animals have problems, sometimes followed by death, due to the high toxicity of the main toxin, crotoxin. Several methods tested to detoxify this toxin often resulted in decreased immunogenicity. Gamma irradiation has proved to be a successful method for crotoxin detoxification without loss of immunogenicity. We have studied the biodistribution of 2 kGy 60Co irradiated crotoxin (iCTX) in mouse tissues. We used both 125I-labeled iCTX or its detection by a specific immunohistochemistry assay (IHA). Both approaches showed similar early excretion of toxins by the kidneys. Higher iCTX uptake was seen in spleen and liver, which are rich in immune responder cells. In contrast to previous reports concerning native crotoxin (nCTX), we failed to detect iCTX in the neuromuscular junction, but both toxins were found on the kidney tubular cell surface, with rapid excretion that was more intense for iCTX. Kupffer cells and splenocyte macrophages presented IHA staining, as shown by the increased uptake of 125I toxin by these organs. No staining was observed in the brain, lung or heart, which also showed very low 125I counts. Allied to reduced toxicity, irradiation induced early endocytosis of crotoxin by phagocytic cells, improving antigen processing.

Irradiation of Crotalus durissus terrificus crotoxin with 60Co gamma-rays induces its uptake by macrophages through scavenger receptors.

PURPOSE: To investigate the action of 2 kGy 60Co gamma-rays on crotoxin and its favoured uptake through scavenger receptor (ScvR) mouse peritoneal macrophages. MATERIALS AND METHODS: Native or irradiated crotoxin (iCTX) (50 microg/ml) dosed with 2 kGy 60Co gamma-rays (dose-rate 540 Gy/h) were offered to mouse peritoneal macrophages; their uptake was evaluated by immunohistochemistry and quantitative in situ ELISA. Receptors recognizing irradiated crotoxin were evaluated with specific ScvR blockers (Probucol and dextran sulphate) or with non-specific blocking using foetal calf serum (FCS). RESULTS: Immunohistochemical assays revealed more deeply staining intracytoplasmic vacuoles in macrophages incubated with iCTX. Using in situ ELISA with ScvR specific blockers, it was shown that the increased uptake of iCTX was blocked by Probucol or dextran sulphate, but not by FCS. On the other hand, the uptake of native crotoxin was decreased by FCS, but not affected by ScvR blockers. The morphology and viability of macrophages were preserved during the experiments. CONCLUSIONS: It is concluded that 60Co gamma-rays probably induced oxidative changes in crotoxin, driving this toxin towards ScvR mouse peritoneal macrophages. This suggests a different in vivo route of iCTX away from toxic neural sites by a preferential and rapid internalization and processing by macrophages, leading to the induction of a better immune response.

Phase I study of VRCTC-310, a purified phospholipase A2 purified from snake venom, in patients with refractory cancer: safety and pharmacokinetic data.

A phase I study was performed to evaluate the maximum tolerated dose (MTD), safety profile and pharmacokinetic data with VRCTC-310, a natural product derived from purified snake venom fractions, with phospholipase A2 activity and inhibitory effects against human and murine tumor cell lines. Fifteen patients with refractory malignancies were entered after providing written informed consent. VRCTC-310 was administered as an intramuscular injection daily for 30 consecutive days. Doses were escalated from 0.0025 to 0.023 mg/kg. Toxicities included local pain at the injection site, eosinophilia, reversible diplopia and palpebral ptosis. Dose escalation was stopped at 0.023 mg/kg, when two patients had developed anaphylactoid reactions. Both cases had high VRCTC-310-specific IgG by EIA. MTD was 0.017 mg/kg and the recommended dose for phase II studies is 0.017 mg/kg. Stabilization was found in six patients.

Serum kinetics of crotoxin from Crotalus durissus terrificus venom in mice: evidence for a rapid clearance.

We report on an ELISA for the detection of crotoxin with a detection limit of 1-3 pg/ml of sample. Cross-reactivity with other animal venoms occurred only at concentrations above 1 microgram/ml. Serum kinetics of crotoxin were investigated in BALB/c mice after a single 10 micrograms s.c. dose of venom obtained from Crotalus durissus terrificus. Crotoxin levels were 254 +/- 141 ng/ml serum (X +/- S.E.) 15 min after venom injection, 3.9 +/- 0.5 ng/ml serum at 30 min and undetectable thereafter. The rapid clearance of crotoxin from the serum suggests that the test may be unsuitable for the clinical management of envenomation victims.