Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation.

The Spike of SARS-CoV-2 recognizes a transmembrane protease, angiotensin-converting enzyme 2 (ACE2), on host cells to initiate infection. Soluble derivatives of ACE2, in which Spike affinity is enhanced and the protein is fused to Fc of an immunoglobulin, are potent decoy receptors that reduce disease in animal models of COVID-19. Mutations were introduced into an ACE2 decoy receptor, including adding custom N-glycosylation sites and a cavity-filling substitution together with Fc modifications, which increased the decoy's catalytic activity and provided small to moderate enhancements of pharmacokinetics following intravenous and subcutaneous administration in humanized FcRn mice. Most prominently, sialylation of native glycans increases exposures by orders of magnitude, and the optimized decoy is therapeutically efficacious in a mouse COVID-19 model. Ultimately, an engineered and highly sialylated decoy receptor produced using methods suitable for manufacture of representative drug substance has high exposure with a 5- to 9-day half-life. Finally, peptide epitopes at mutated sites in the decoys generally have low binding to common HLA class II alleles and the predicted immunogenicity risk is low. Overall, glycosylation is a critical molecular attribute of ACE2 decoy receptors and modifications that combine tighter blocking of Spike with enhanced pharmacokinetics elevate this class of molecules as viable drug candidates.

Phase 1 Safety, Pharmacokinetics, and Fluorescence Imaging Study of Tozuleristide (BLZ-100) in Adults With Newly Diagnosed or Recurrent Gliomas.

BACKGROUND: Fluorescence-guided surgery (FGS) can improve extent of resection in gliomas. Tozuleristide (BLZ-100), a near-infrared imaging agent composed of the peptide chlorotoxin and a near-infrared fluorophore indocyanine green, is a candidate molecule for FGS of glioma and other tumor types. OBJECTIVE: To perform a phase 1 dose-escalation study to characterize the safety, pharmacokinetics, and fluorescence imaging of tozuleristide in adults with suspected glioma. METHODS: Patients received a single intravenous dose of tozuleristide 3 to 29 h before surgery. Fluorescence images of tumor and cavity in Situ before and after resection and of excised tissue ex Vivo were acquired, along with safety and pharmacokinetic measures. RESULTS: A total of 17 subjects received doses between 3 and 30 mg. No dose-limiting toxicity was observed, and no reported adverse events were considered related to tozuleristide. At doses of 9 mg and above, the terminal serum half-life for tozuleristide was approximately 30 min. Fluorescence signal was detected in both high- and low-grade glial tumors, with high-grade tumors generally showing greater fluorescence intensity compared to lower grade tumors. In high-grade tumors, signal intensity increased with increased dose levels of tozuleristide, regardless of the time of dosing relative to surgery. CONCLUSION: These results support the safety of tozuleristide at doses up to 30 mg and suggest that tozuleristide imaging may be useful for FGS of gliomas.

Pharmacokinetics and pharmacodynamics of pegylated interferon lambda-1 in cynomolgus monkeys.

Type III lambda interferons (IFNs) have activity similar to type I IFNs, but a more restricted receptor distribution. A pegylated human IFN lambda-1 (pegIFNλ) is under development for chronic hepatitis C. Induction of receptor signaling (STAT1 phosphorylation) and expression of interferon-stimulated genes (ISGs) by pegIFNλ were assessed in, respectively, cynomolgus monkey leukocyte subsets and hepatocytes stimulated in vitro. ISG induction by pegIFNλ or IFNα was also assessed in peripheral leukocytes and liver biopsies after single and repeat (x3) dosing of pegIFNλ (0.03, 0.3, 3.0 mg/kg) or unpegylated IFNα-2b (10(7) IU/kg). Single-dose pharmacokinetics of pegIFNλ were evaluated. Strong ISG induction occurred in cultured hepatocytes and liver biopsies with both pegIFNλ and IFNα. However, STAT1 phosphorylation, MHC class 1 upregulation, and ISG induction in leukocytes only occurred with IFNα. Serum neopterin was unaffected by pegIFNλ; however, ß-2-microglobulin was elevated at all doses. The terminal half-life of pegIFNλ was 23 h with a 59 mL/kg volume of distribution, consistent with other pegylated IFNs. Serum exposure was dose-proportional across the dosing range. These data demonstrate the suitability of cynomolgus monkeys for the preclinical evaluation of pegIFNλ. Additionally, the absence of pegIFNλ pharmacologic activity in leukocytes is consistent with its low receptor expression in blood.

Sex differences in (+)-amphetamine- and (+)-methamphetamine-induced behavioral response in male and female Sprague-Dawley rats.

(+)-Methamphetamine (METH) and (+)-amphetamine (AMP) are structurally similar drugs that are reported to induce similar pharmacological effects in rats of the same sex. Because pharmacokinetic data suggest female rats should be more affected than males, the current studies sought to test the hypothesis that the behavioral and temporal actions of METH and AMP should be greater in female Sprague-Dawley rats than in males. Using a dosing regimen designed to reduce the possibility of tolerance and sensitization, rats were administered 1.0 and 3.0 mg/kg intravenous drug doses. Distance traveled, rearing events and focal stereotypies (e.g., head weaving, sniffing) were measured. Female rats traveled significantly greater distances and displayed a greater number of rearing events than males after both doses. Analysis of stereotypy ratings after 3.0 mg/kg revealed that focal stereotypies were more pronounced and lasted longer in females. The second study compared the potencies of METH and AMP in inducing locomotor activity and focal stereotypies in each sex. No differences in potency were found when METH and AMP effects were compared within males or females. In summary, these studies showed female rats displayed greater and longer-lasting locomotor activity and more stereotypic behaviors, supporting earlier evidence of significant sexual dimorphism in pharmacokinetics.

Monoclonal IgG affinity and treatment time alters antagonism of (+)-methamphetamine effects in rats.

The roles of monoclonal antibody affinity and treatment time of (+)-methamphetamine-induced pharmacological effects in rats were studied using two anti-(+)-methamphetamine monoclonal antibodies. These studies tested the preclinical protective effects of monoclonal antibody antagonists in (+)-methamphetamine overdose and pretreatment scenarios. The higher affinity antibody (mAb6H4; KD=11 nM for (+)-methamphetamine) more effectively antagonized (+)-methamphetamine-induced behavioral effects (distance and rearing) than the low affinity antibody (designated mAb6H8; KD=250 nM) and had a longer duration of action. Both antibodies more effectively reduced (+)-methamphetamine effects in the overdose model than in the pretreatment model. (+)-Methamphetamine pharmacokinetic studies showed the mAb6H4 significantly reduced brain concentrations over time in both models. However, while mAb6H4 immediately reduced brain concentrations in the overdose model, it did not prevent the initial distribution of (+)-methamphetamine into the brain in the pretreatment model. Thus, anti-(+)-methamphetamine monoclonal antibody affinity and administration time (relative to (+)-methamphetamine dosing) are critical determinants of therapeutic success.

Use of anti-(+)-methamphetamine monoclonal antibody to significantly alter (+)-methamphetamine and (+)-amphetamine disposition in rats.

These studies examined the effects of a high-affinity anti-(+)-methamphetamine monoclonal antibody (mAb; KD = 11 nM) on (+)-methamphetamine [(+)-METH] and (+)-amphetamine [(+)-AMP] serum and tissue disposition and serum protein binding following i.v. (+)-METH administration. Male Sprague-Dawley rats were pretreated with a buffer solution (control rats) or with anti-(+)-METH mAb [equimolar in binding sites to the (+)-METH dose]. The next day, both groups received a 1 mg/kg i.v. (+)-METH dose. At various time points after (+)-METH administration, rats were sacrificed (n = 3 per time point), and serum and tissues were collected. (+)-METH serum protein binding was increased from approximately 5% in controls to approximately 88 to 99% in the mAb-treated rats. The (+)-METH area under the concentration versus time curves from 0 to 4.5 h (AUC0-4.5 h) in mAb-treated rats showed an increase of >6600% for serum and a decrease of >60% for brain, compared with buffer-treated controls. Differential effects of anti-METH mAb on (+)-METH concentrations were observed in other tissues. For example, in the liver, anti-(+)-METH mAb caused significant increases in (+)-METH concentrations. The AUC0-4.5 h for (+)-AMP, a pharmacologically active metabolite, was decreased by approximately 50% in all tissues examined. These data show that pretreatment with an anti-(+)-METH mAb can significantly alter the disposition of (+)-METH and (+)-AMP in rats. Since the mAb has no significant cross-reactivity with (+)-AMP, the data suggest that the mAb reduced (+)-METH metabolic clearance through high-affinity binding to (+)-METH. Finally, rapidly equilibrating tissues, like the brain, appear to be preferentially protected by the mAb.

Pharmacodynamic mechanisms of monoclonal antibody-based antagonism of (+)-methamphetamine in rats.

Our studies examined pharmacokinetic mechanisms involved in high-affinity (K(d) approximately 11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg/kg i.v. (+)-methamphetamine dose 30 min after administration. The monoclonal antibody antagonized locomotor effects due to 0.3 and 1 mg/kg (+)-methamphetamine. In contrast, monoclonal antibody treatment increased locomotor activity due to 3 mg/kg (+)-methamphetamine. We also investigated the serum and brain pharmacokinetics of (+)-methamphetamine without and with the monoclonal antibody. Rats received (+)-methamphetamine (1 mg/kg i.v.) followed by saline or monoclonal antibody treatment at 30 min. The monoclonal antibody significantly increased serum methamphetamine concentrations and significantly decreased brain methamphetamine concentrations. These data indicate that anti-(+)-methamphetamine monoclonal antibody-induced pharmacodynamics are complex, but are related to time-dependent changes in (+)-methamphetamine brain distribution.