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BLZ-100 (tozuleristide) is an intraoperative fluorescent imaging agent that selectively detects malignant tissue and can be used in real time to guide tumor resection. The purpose of this study was to assess the safety, tolerability, and pharmacokinetics of BLZ-100 and to explore the pharmacodynamics of fluorescence imaging of skin tumors. In this first-in-human study, BLZ-100 was administered intravenously to 21 adult patients 2 days before excising known or suspected skin cancers. Doses were 1, 3, 6, 12, and 18 mg, with 3–6 patients/cohort. Fluorescence imaging was conducted before and up to 48 h after dosing. BLZ-100 was well tolerated. There were no serious adverse events, deaths, or discontinuations due to adverse events, and no maximum tolerated dose (MTD) was identified. Headache (n = 2) and nausea (n = 2) were the only BLZ-100 treatment-related adverse events reported for >1 patient. Median time to maximal serum concentration was

On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads.

Objective To determine the utility of skin biopsies as a biomarker of disease severity in subjects with amyloid neuropathy. Methods Five groups of patients were studied: (1) transthyretin (TTR) familial amyloidotic polyneuropathy (FAP; n = 20), (2) TTR mutation carriers without peripheral neuropathy (TTR-noPN; n = 10), (3) healthy controls (n = 20), (4) diabetic neuropathy disease controls (n = 20), and (5) patients with light-chain (AL) amyloid (n = 2). All subjects underwent neurological examination and 3mm skin biopsies. Sections were stained with anti-PGP9.5, anti-TTR, and Congo red. Intraepidermal (IENFD), sweat gland (SGNFD), and pilomotor nerve fiber densities (PMNFD) were measured. Correlations between the amount of amyloid present (amyloid burden), fiber subtype, and Neuropathy Impairment Score in the Lower Limbs (NIS-LL) were evaluated. Results IENFD, SGNFD, and PMNFD were all significantly reduced in TTR-FAP patients versus healthy controls, whereas TTR-noPN subjects had intermediate reductions. Lower nerve fiber densities were associated with NIS-LL (p

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. Refereed/Peer-reviewed

We compared patterns of intraepidermal nerve fibers and mechanoreceptors from affected and unaffected plantar skin from patients with pachyonychia congenita (PC) and control subjects. Plantar biopsies from 10 genetically confirmed patients with PC (with a mutation in KRT6A) were performed at the ball of the foot (affected skin) and the arch (unaffected) and were compared to biopsies from corresponding locations in 10 control subjects. Tissue was processed to visualize intraepidermal nerve fibers (IENF) (PGP9.5), subsets of IENF (CGRP, substance P, tyrosine hydroxylase), myelinated nerve fiber (neurofilament H, NFH), blood vessels (CD31), Meissner corpuscles, and Merkel cells (MCs). Structures were quantified using stereology or validated quantification methods. We observed that PC-affected plantar skin had significantly lower sweat gland innervation (sweat gland nerve fiber density) and reduced numbers of Meissner corpuscles compared to PC-unaffected or anatomically matched control skin. In contrast, Merkel cell densities and blood vessel counts were higher in PC-affected skin compared to either control or PC-unaffected skin. There were no differences in myelinated nerve fiber densities, SP, or CGRP between the groups. Pressure pain thresholds in PC-affected skin were lower compared to PC-unaffected and anatomically matched control skin. Additionally, MC densities in callused plantar skin from healthy runners with callus and one subject with a nonpainful palmoplantar keratoderma (AQP5 mutation) were similar to PC-unaffected and control skin consistent with callus alone not being sufficient to increase MC number. These findings suggest that alterations in PC extend beyond keratinocytes and may provide strategies to study neuropathic pain in PC.

Background Overexpression or administration of interleukin 31 (IL-31) has been shown to induce a profound itch response in mice and dogs. The chronic pruritus observed in mouse IL-31 transgenic mice results in the development of skin lesions and alopecia through excoriation from excessive scratching, a condition similar to that observed in patients with atopic dermatitis (AD). Objective To test whether IL-31 induces pruritus in non-human primates and, if so, whether treatment with an anti-IL-31 neutralizing monoclonal antibody (mAb) can block the response. Methods A series of studies was conducted in cynomolgus monkeys to evaluate the itch response to recombinant cynomolgus IL-31 (cIL-31) administration. Three routes of cIL-31 administration (intravenous, intradermal, and subcutaneous) were evaluated. Subcutaneous treatment with a humanized anti-human IL-31 mAb cross-reactive to cIL-31 was subsequently tested for its ability to block the response to intradermal cIL-31 administration. Results Each route of cIL-31 delivery elicited a scratching response immediately after cIL-31 administration and lasted at least 3 h. Treatment with the IL-31 mAb inhibited the cIL-31-mediated scratching response in a dose-dependent manner. Conclusion These results demonstrate that an IL-31 mAb can inhibit IL-31-mediated pruritus in vivo, and could be an effective therapy for pruritic skin conditions like AD where IL-31 upregulation may play a role.

BLZ-100 is a single intravenous use, fluorescent imaging agent that labels tumor tissue to enable more complete and precise surgical resection. It is composed of a chlorotoxin peptide covalently bound to the near-infrared fluorophore indocyanine green. BLZ-100 is in clinical development for intraoperative visualization of human tumors. The nonclinical safety and pharmacokinetic (PK) profile of BLZ-100 was evaluated in mice, rats, canines, and nonhuman primates (NHP). Single bolus intravenous administration of BLZ-100 was well tolerated, and no adverse changes were observed in cardiovascular safety pharmacology, PK, and toxicology studies in rats and NHP. The single-dose no-observed-adverse-effect-levels (NOAELs) were 7 mg (28 mg/kg) in rats and 60 mg (20 mg/kg) in NHP, corresponding to peak concentration values of 89 400 and 436 000 ng/mL and area-under-the-curve exposure values of 130 000 and 1 240 000 h·ng/mL, respectively. Based on a human imaging dose of 3 mg, dose safety margins are >100 for rat and monkey. BLZ-100 produced hypersensitivity reactions in canine imaging studies (lethargy, pruritus, swollen muzzle, etc). The severity of the reactions was not dose related. In a follow-up study in dogs, plasma histamine concentrations were increased 5 to 60 minutes after BLZ-100 injection; this coincided with signs of hypersensitivity, supporting the conclusion that the reactions were histamine based. Hypersensitivity reactions were not observed in other species or in BLZ-100 human clinical studies conducted to date. The combined imaging, safety pharmacology, PK, and toxicology studies contributed to an extensive initial nonclinical profile for BLZ-100, supporting first-in-human clinical trials.

To determine the utility of skin biopsies as a biomarker of disease severity in subjects with amyloid neuropathy.Five groups of patients were studied: (1) transthyretin (TTR) familial amyloidotic polyneuropathy (FAP; n = 20), (2) TTR mutation carriers without peripheral neuropathy (TTR-noPN; n = 10), (3) healthy controls (n = 20), (4) diabetic neuropathy disease controls (n = 20), and (5) patients with light-chain (AL) amyloid (n = 2). All subjects underwent neurological examination and 3mm skin biopsies. Sections were stained with anti-PGP9.5, anti-TTR, and Congo red. Intraepidermal (IENFD), sweat gland (SGNFD), and pilomotor nerve fiber densities (PMNFD) were measured. Correlations between the amount of amyloid present (amyloid burden), fiber subtype, and Neuropathy Impairment Score in the Lower Limbs (NIS-LL) were evaluated.IENFD, SGNFD, and PMNFD were all significantly reduced in TTR-FAP patients versus healthy controls, whereas TTR-noPN subjects had intermediate reductions. Lower nerve fiber densities were associated with NIS-LL (p 0.001). Congo red staining revealed brilliant red amyloid deposits confirmed by apple-green birefringence within dermal collagen, sweat glands, and arrector pili that engulfed axons. The diagnostic sensitivity and specificity to detect amyloid in skin were 70% and 100%. Both AL amyloidosis and 2 of 10 TTR-noPN subjects were Congo red-positive. Amyloid burden correlated with IENFD (r = -0.63), SGNFD (r = -0.67), PMNFD (r = -0.50), and NIS-LL (r = -0.57). Wild-type TTR staining was less prominent in TTR-FAP patients.Cutaneous amyloid was detected in 70% of TTR-FAP and 20% of TTR-noPN subjects. Amyloid burden correlated strongly with reductions in IENFD, SGNFD, PMNFD, and NIS-LL. Skin is an attractive tissue to establish an amyloid diagnosis, and amyloid burden has potential as a biomarker to detect treatment effect in TTR-FAP drug trials. Ann Neurol 2017;82:44-56.

Blood-borne RNA circulating in association with autoantibodies is a potent stimulator of interferon production and immune system activation. RSLV-132 is a novel fully human biologic Fc fusion protein that is comprised of human RNase fused to the Fc domain of human IgG1. The drug is designed to remain in circulation and digest extracellular RNA with the aim of preventing activation of the immune system via Toll-like receptors and the interferon pathway. The present study describes the first clinical study of nuclease therapy in 32 subjects with systemic lupus erythematosus. The drug was well tolerated with a very favorable safety profile. The approximately 19-day serum half-life potentially supports once monthly dosing. There were no subjects in the study that developed anti-RSLV-132 antibodies. Decreases in B-cell activating factor correlated with decreases in disease activity in a subset of patients.

e14562Background: Tozuleristide (BLZ-100) is a fluorescent imaging agent intended to highlight tumor tissue in patients undergoing surgical tumor resection. The pharmacokinetics (PK) of BLZ-100 in ...

Interferon lambda 1 (IFN-lambda1) is a type III IFN that produces intracellular responses similar to those of IFN-alpha but in fewer cell types because of differences in the receptor distribution pattern, and this could potentially result in an improved safety profile. This was an open-label three-part study of patients with chronic hepatitis C virus (HCV) genotype 1 infection. Part 1 evaluated single-agent pegylated interferon lambda (PEG-IFN-lambda) at 1.5 or 3.0 microg/kg administered every 2 weeks or weekly for 4 weeks in patients who had relapsed after previous IFN-alpha-based treatment. Part 2 evaluated weekly doses of PEG-IFN-lambda ranging from 0.5 to 2.25 microg/kg in combination with ribavirin (RBV) for 4 weeks in treatment-relapse patients. Part 3 evaluated weekly PEG-IFN-lambda at 1.5 microg/kg in combination with RBV for 4 weeks in treatment-naive patients. Fifty-six patients were enrolled: 24 patients in part 1, 25 patients in part 2, and 7 patients in part 3. Antiviral activity was observed at all PEG-IFN-lambda dose levels (from 0.5 to 3.0 microg/kg). Two of seven treatment-naive patients (29%) achieved rapid virological response. Treatment was well tolerated with minimal flu-like symptoms and no significant hematologic changes other than RBV-associated decreases in hemoglobin. The most common adverse events were fatigue (29%), nausea (12%), and myalgia (11%). Six patients experienced increases in aminotransferases that met protocol-defined criteria for dose-limiting toxicity (DLT) or temporarily holding therapy with PEG-IFN-lambda. Most DLT occurred in patients with high PEG-IFN-lambda exposure.Weekly PEG-IFN-lambda with or without daily RBV for 4 weeks is well tolerated with minimal adverse events and hematologic effects and is associated with clear antiviral activity across a broad range of doses in patients with chronic HCV.

We thank Eglin et al for their thoughtful commentary on our manuscript, in which we undertook the first detailed neurological assessment of persistent symptoms following non-freezing cold injury. Whilst we stress that sensory neuropathy is one core feature of NFCI we do not want to give the impression that vascular factors may not play a role. In fact, given the intimate relationship between peripheral neurons and blood vessels in the neurovascular unit, it would be a surprise if vascular changes were not observed.

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.

Background Despite the positive impact of targeted therapies on metastatic renal cell carcinoma (mRCC), durable responses are infrequent and an unmet need exists for novel therapies with distinct mechanisms of action. We investigated the combination of recombinant Interleukin 21 (IL-21), a cytokine with unique immunostimulatory properties, plus sorafenib, a VEGFR tyrosine kinase inhibitor. Methods In this phase 1/2 study, 52 mRCC patients received outpatient treatment with oral sorafenib 400 mg twice daily plus intravenous IL-21 (10–50 mcg/kg) on days 1–5 and 15–19 of each 7-week treatment course. The safety, antitumor activity, pharmacokinetic and pharmacodynamic effects of the combination were evaluated. Results In phase 1 (n = 19), the maximum tolerated dose for IL-21 with the standard dose of sorafenib was determined to be 30 mcg/kg/day; grade 3 skin rash was the only dose-limiting toxicity. In phase 2, 33 previously-treated patients tolerated the combination therapy well with appropriate dose reductions; toxicities were mostly grade 1 or 2. The objective response rate was 21% and disease control rate was 82%. Two patients have durable responses that are ongoing, despite cessation of both IL-21 and sorafenib, at 41+ and 30+ months, respectively. The median progression-free survival in phase 2 was 5.6 months. The pharmacokinetic and pharmacodynamic properties of IL-21 appeared to be preserved in the presence of sorafenib. Conclusion IL-21 plus sorafenib has antitumor activity and acceptable safety in previously treated mRCC patients. IL-21 may represent a suitable immunotherapy in further exploration of combination strategies in mRCC. Trial registration ClinicalTrials.gov Identifier: NCT00389285

The goal of these studies was to determine if chronic (+)methamphetamine ((+)METH) administration affects the production of anti-(+)METH antibodies during active immunization of rats. Active immunization for the treatment of chronic drug abuse has been proposed for drugs such as cocaine and nicotine. However, studies have not adequately addressed whether continual drug use during treatment would affect the development of an immune response. For the current studies, male Sprague–Dawley rats were immunized with either keyhole limpet hemocyanin (KLH; control group) or a (+)METH hapten ((+)METH with a six carbon spacer group at the para position of the ring structure)–KLH conjugate. The (+)METH–KLH animals were further divided into two groups. One group was immunized with no subsequent administration of (+)METH, while the other group was immunized and repeatedly challenged (twice a week throughout the study) with an i.p. dose of 3 mg/kg (+)METH. The results showed that the two groups of (+)METH–KLH immunized rats developed and maintained anti-(+)METH antibody titers. The anti-(+)METH immune responses of the two groups were not statistically different (P

Interleukin-21 (IL-21), a pleiotropic immunostimulatory type I cytokine, has anticancer effects in animal models. Preclinical studies designed to assess the safety of recombinant human IL-21 (rIL-21) for use in phase I oncology studies are described. The rIL-21 (≤3.0 mg/kg per dose) was given intravenously to cynomolgus monkeys ( Macaca fascicularis) once daily for 5 days, followed by 9 nondosing days (1 cycle) for ≤4 cycles. The rIL-21 pharmacokinetics was dose-dependent. Accumulation was not observed after repeated dosing, consistent with the short elimination half-life ( t1/2,λz; 0.4-0.8 hours). Safety findings included cyclical anemia and thrombocytopenia, clinical pathology changes consistent with acute-phase response, leukocyte infiltrates in hepatic sinusoids, and sporadic serum transaminase elevations (typically

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, β-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.

Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.

Binding of immune complexes to cellular FcγRs can promote cell activation and inflammation. In previous studies, a recombinant human (rh) soluble FcγR, rh-FcγRIA (CD64A), was shown to block inflammation in passive transfer models of immune complex-mediated disease. To assess whether rh-FcγRIA could block inflammation in a T cell- and B cell-dependent model of immune complex-mediated disease, the efficacy of rh-FcγRIA in collagen-induced arthritis was evaluated. Mice with established arthritis were treated with a single s.c. injection of rh-FcγRIA (0.2–2.0 mg/dose) given every other day for 11 days. Relative to mice injected with vehicle alone, mice treated with rh-FcγRIA exhibited lower serum concentrations of IL-6, anti-type II collagen Abs, and total IgG2a. These changes were correlated with lower levels of paw swelling and joint damage in the rh-FcγRIA-treated mice and occurred in the presence of a significant murine Ab response to rh-FcγRIA. Comparison of the serum rh-FcγRIA concentration vs time profiles for rh-FcγRIA administered at two dose levels by i.v. and s.c. injection revealed that the bioavailabilty of s.c. administered rh-FcγRIA was 27–37%. Taken together, these data show that rh-FcγRIA is an effective inhibitor of inflammation in a model of established arthritis in mice.

TPS9084 Background: BLZ-100 is an intraoperative, fluorescent imaging agent designed to specifically label malignant tissue and enable more complete surgical resection of tumor tissue. BLZ-100 achi...

(+)-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.

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.

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 of6600% for serum and a decrease of60% 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.