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.

TPP1 Delivery to Lysosomes with Extracellular Vesicles and their Enhanced Brain Distribution in the Animal Model of Batten Disease.

Extracellular vesicles (EVs) are promising natural nanocarriers for delivery of various types of therapeutics. Earlier engineered EV-based formulations for neurodegenerative diseases and cancer are reported. Herein, the use of macrophage-derived EVs for brain delivery of a soluble lysosomal enzyme tripeptidyl peptidase-1, TPP1, to treat a lysosomal storage disorder, Neuronal Ceroid Lipofuscinoses 2 (CLN2) or Batten disease, is investigated. TPP1 is loaded into EVs using two methods: i) transfection of parental EV-producing macrophages with TPP1-encoding plasmid DNA (pDNA) or ii) incorporation therapeutic protein TPP1 into naive empty EVs. For the former approach, EVs released by pretransfected macrophages contain the active enzyme and TPP1-encoding pDNA. To achieve high loading efficiency by the latter approach, sonication or permeabilization of EV membranes with saponin is utilized. Both methods provide proficient incorporation of functional TPP1 into EVs (EV-TPP1). EVs significantly increase stability of TPP1 against protease degradation and provide efficient TPP1 delivery to target cells in in vitro model of CLN2. The majority of EV-TPP1 (≈70%) is delivered to target organelles, lysosomes. Finally, a robust brain accumulation of EV carriers and increased lifespan is recorded in late-infantile neuronal ceroid lipofuscinosis (LINCL) mouse model following intraperitoneal administration of EV-TPP1.

Nonclinical evaluation of CNS-administered TPP1 enzyme replacement in canine CLN2 neuronal ceroid lipofuscinosis.

The CLN2 form of neuronal ceroid lipofuscinosis, a type of Batten disease, is a lysosomal storage disorder caused by a deficiency of the enzyme tripeptidyl peptidase-1 (TPP1). Patients exhibit progressive neurodegeneration and loss of motor, cognitive, and visual functions, leading to death by the early teenage years. TPP1-null Dachshunds recapitulate human CLN2 disease. To characterize the safety and pharmacology of recombinant human (rh) TPP1 administration to the cerebrospinal fluid (CSF) as a potential enzyme replacement therapy (ERT) for CLN2 disease, TPP1-null and wild-type (WT) Dachshunds were given repeated intracerebroventricular (ICV) infusions and the pharmacokinetic (PK) profile, central nervous system (CNS) distribution, and safety were evaluated. TPP1-null animals and WT controls received 4 or 16mg of rhTPP1 or artificial cerebrospinal fluid (aCSF) vehicle every other week. Elevated CSF TPP1 concentrations were observed for 2-3 days after the first ICV infusion and were approximately 1000-fold higher than plasma levels at the same time points. Anti-rhTPP1 antibodies were detected in CSF and plasma after repeat rhTPP1 administration, with titers generally higher in TPP1-null than in WT animals. Widespread brain distribution of rhTPP1 was observed after chronic administration. Expected histological changes were present due to the CNS delivery catheters and were similar in rhTPP1 and vehicle-treated animals, regardless of genotype. Neuropathological evaluation demonstrated the clearance of lysosomal storage, preservation of neuronal morphology, and reduction in brain inflammation with treatment. This study demonstrates the favorable safety and pharmacology profile of rhTPP1 ERT administered directly to the CNS and supports clinical evaluation in patients with CLN2 disease.

Recombinant human tripeptidyl peptidase-1 infusion to the monkey CNS: safety, pharmacokinetics, and distribution.

CLN2 disease is caused by deficiency in tripeptidyl peptidase-1 (TPP1), leading to neurodegeneration and death. The safety, pharmacokinetics (PK), and CNS distribution of recombinant human TPP1 (rhTPP1) were characterized following a single intracerebroventricular (ICV) or intrathecal-lumbar (IT-L) infusion to cynomolgus monkeys. Animals received 0, 5, 14, or 20mg rhTPP1, ICV, or 14 mg IT-L, in artificial cerebrospinal fluid (aCSF) vehicle. Plasma and CSF were collected for PK analysis. Necropsies occurred at 3, 7, and 14 days post-infusion. CNS tissues were sampled for rhTPP1 distribution. TPP1 infusion was well tolerated and without effect on clinical observations or ECG. A mild increase in CSF white blood cells (WBCs) was detected transiently after ICV infusion. Isolated histological changes related to catheter placement and infusion were observed in ICV treated animals, including vehicle controls. The CSF and plasma exposure profiles were equivalent between animals that received an ICV or IT-L infusion. TPP1 levels peaked at the end of infusion, at which point the enzyme was present in plasma at 0.3% to 0.5% of CSF levels. TPP1 was detected in brain tissues with half-lives of 3-14 days. CNS distribution between ICV and IT-L administration was similar, although ICV resulted in distribution to deep brain structures including the thalamus, midbrain, and striatum. Direct CNS infusion of rhTPP1 was well tolerated with no drug related safety findings. The favorable nonclinical profile of ICV rhTPP1 supports the treatment of CLN2 by direct administration to the CNS.

Systemic administration of tripeptidyl peptidase I in a mouse model of late infantile neuronal ceroid lipofuscinosis: effect of glycan modification.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a recessive genetic disease of childhood caused by deficiencies in the lysosomal protease tripeptidyl peptidase I (TPP1). Disease is characterized by progressive and extensive neuronal death. One hurdle towards development of enzyme replacement therapy is delivery of TPP1 to the brain. In this study, we evaluated the effect of modifying N-linked glycans on recombinant human TPP1 on its pharmacokinetic properties after administration via tail vein injection to a mouse model of LINCL. Unmodified TPP1 exhibited a dose-dependent serum half-life of 12 min (0.12 mg) to 45 min (2 mg). Deglycosylation or modification using sodium metaperiodate oxidation and reduction with sodium borohydride increased the circulatory half-life but did not improve targeting to the brain compared to unmodified TPP1. Analysis of liver, brain, spleen, kidney and lung demonstrated that for all preparations, >95% of the recovered activity was in the liver. Interestingly, administration of a single 2 mg dose (80 mg/kg) of unmodified TPP1 resulted in ∼10% of wild-type activity in brain. This suggests that systemic administration of unmodified recombinant enzyme merits further exploration as a potential therapy for LINCL.