A phase I study of the antibody drug conjugate ASG-5ME, an SLC44A4-targeting antibody carrying auristatin E, in metastatic castration-resistant prostate cancer.

Background Antibody drug conjugates (ADC) offer the potential of maximizing efficacy while minimizing systemic toxicity. ASG-5ME, an SLC44A4-targeting antibody carrying monomethyl auristatin E (MMAE), a microtubule-disrupting agent, was investigated in men with metastatic castration resistant prostate cancer. Methods The primary objective of this phase I study was to determine maximum tolerated dose (MTD) and recommended phase II dose. Secondary objectives were safety, antitumor activity, pharmacokinetic properties, immunogenicity, and the detection of SLC44A4 on circulating tumor cells. Patients (pts) were treated among 7 dose levels every 21 days. A dose expansion phase enrolled 20 additional pts. at the MTD. Results Twenty-six and 20 pts. were treated in dose escalation and dose expansion cohorts respectively. The MTD was 2.7 mg/kg. Dose-limiting toxicities occurred in 4 pts.: grade 3 fatigue (n = 1); grade 3 abdominal pain, diarrhea and fatigue (n = 1); grade 4 neutropenia and hyponatremia and grade 3 maculopapular rash, constipation and hypoxia (n = 1); grade 3 troponin elevation without cardiac sequelae (n = 1). Fatigue and diarrhea were the most prevalent adverse events (AEs) across all cycles. Two grade 5 AEs occurred in the dose expansion cohort, each after 1 dose: 1 pt. developed grade 3 hyperglycemia, renal insufficiency and leukopenia; 1 pt. developed grade 3 hyperglycemia complicated by bacteremia. Free MMAE levels did not accumulate with repeat dosing. Of evaluable pts., 52% had either stable disease or a partial response. Conclusions Further development of ASG-5ME is not being pursued due to its narrow therapeutic index. Some toxicities were potentially related to on-target effects on normal tissue expressing the SLC44A4 protein. However, other toxicities were consistent with studies of previous MMAE-containing ADCs. Unconjugated MMAE is a less likely etiology based on prior data.

Metabolism of an Oxime-Linked Antibody Drug Conjugate, AGS62P1, and Characterization of Its Identified Metabolite.

AGS62P1 is an antibody drug conjugate (ADC) composed of a human IgG1κ monoclonal antibody against FLT3 (FMS-like tyrosine kinase 3) with a p-acetyl phenylalanine (pAF) residue inserted at position 124 of each heavy chain linked to the proprietary microtubule disrupting agent AGL-0182-30 via an alkoxyamine linker that forms an oxime upon conjugation to the antibody. AGS62P1 is currently in Phase I human clinical trials for acute myelogenous leukemia (AML). The identified primary metabolite of an oxime-linked ADC is presented for the first time. AGS62P1 metabolism was assessed in xenograft tumor-bearing mice and rats treated with the ADC using liquid chromatography and mass spectrometry-based methods described herein. In this study, we identified the metabolite of AGS62P1 as pAF-AGL-0185-30, which contains a fragment resulting from the catabolism of the antibody component of the ADC and hydrolysis of the terminal amide portion of the linker-payload. We demonstrated that the metabolite of AGS62P1 is tolerated in rats above 1.5 mg/kg and above 0.334 mg/kg in cynomolgus monkeys when given as a single dose. Furthermore, we established in vitro that pAF-AGL-0185-30 does not significantly inhibit hERG or cytochrome P450 family enzymes (CYPs).

HPN328, a Trispecific T Cell-activating Protein Construct Targeting DLL3-Expressing Solid Tumors.

Delta-like ligand 3 (DLL3) is expressed in more than 70% of small cell lung cancers (SCLCs) and other neuroendocrine-derived tumor types. SCLC is highly aggressive and limited therapeutic options lead to poor prognosis for patients. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a CD3 binder for T cell engagement, an albumin binder for half-life extension, and a DLL3 binder for tumor cell engagement. In vitro assays, rodent models and non-human primates were used to assess the activity of HPN328. HPN328 induces potent dose-dependent killing of DLL3-expressing SCLC cell lines in vitro concomitant with T cell activation and cytokine release. In an NCI-H82 xenograft model with established tumors, HPN328 treatment led to T cell recruitment and anti-tumor activity. In an immunocompetent mouse model expressing a human CD3ε epitope, mice previously treated with HPN328 withstood tumor rechallenge, demonstrating long-term anti-tumor immunity. When repeat doses were administered to cynomolgus monkeys, HPN328 was well tolerated up to 10 mg/kg. Pharmacodynamic changes, such as transient cytokine elevation, were observed, consistent with the expected mechanism of action of T cell engagers. HPN328 exhibited linear pharmacokinetic in the given dose range with a serum half-life of 78 to 187 hours, supporting weekly or less frequent administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A phase 1/2 clinical trial is currently underway testing safety and efficacy in patients with DLL3 expressing malignancies.

Safety and Efficacy of Engineered Toxin Body MT-3724 in Relapsed or Refractory B-cell Non-Hodgkin's Lymphomas and Diffuse Large B-cell Lymphoma.

MT-3724, a novel engineered toxin body comprised of an anti-CD20 single-chain variable fragment genetically fused to Shiga-like Toxin A subunit, is capable of binding to and internalizing against CD20, inducing cell killing via permanent ribosomal inactivation. This study evaluated MT-3724 in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (r/rNHL). This open-label, multiple-dose phase Ia/b trial included a dose escalation in patients with r/rNHL according to a standard 3+3 design. Primary objectives were to determine the MTD and pharmacokinetics/pharmacodynamics. In a dose expansion study at MTD in serum rituximab-negative patients with diffuse large B-cell lymphoma (DLBCL), primary objectives were safety, tolerability, and pharmacokinetics/pharmacodynamics. Twenty-seven patients enrolled. MTD was 50 µg/kg/dose with 6,000 µg/dose cap. Thirteen patients experienced at least one grade ≥3 treatment-related adverse events; the most common grade ≥3 event was myalgia (11.1%). Two patients (75 µg/kg/dose) experienced grade 2 treatment-related capillary leak syndrome. Overall objective response rate was 21.7%. In serum rituximab-negative patients with DLBCL or composite DLBCL (n = 12), overall response rate was 41.7% (complete response, n = 2; partial response, n = 3). In patients with detectable baseline peripheral B cells, treatment resulted in dose-dependent B-cell depletion. The proportion of patients with anti-drug antibodies (ADA) increased during treatment and the majority appeared to be neutralizing based on an in vitro assay; nevertheless, tumor regression and responses were observed. MT-3724 demonstrated efficacy at the MTD in this population of previously treated patients with r/rDLBCL, with mild-to-moderate immunogenic safety events. Significance: This work describes the safety and efficacy of a new pharmaceutical pathway that could provide a treatment option for a subset of patients with a critical unmet therapeutic need. The study drug, MT-3724, is capable of targeting B-cell lymphomas via a unique, potent cell-killing mechanism that appears to be promising.

Phase I Trials of Anti-ENPP3 Antibody-Drug Conjugates in Advanced Refractory Renal Cell Carcinomas.

Purpose: To determine the safety, pharmacokinetics, and recommended phase II dose of an antibody-drug conjugate (ADC) targeting ectonucleotide phosphodiesterases-pyrophosphatase 3 (ENPP3) conjugated to monomethyl auristatin F (MMAF) in subjects with advanced metastatic renal cell carcinoma (mRCC).Patients and Methods: Two phase I studies were conducted sequentially with 2 ADCs considered equivalent, hybridoma-derived AGS-16M8F and Chinese hamster ovary-derived AGS-16C3F. AGS-16M8F was administered intravenously every 3 weeks at 5 dose levels ranging from 0.6 to 4.8 mg/kg until unacceptable toxicity or progression. The study was terminated before reaching the MTD. A second study with AGS-16C3F started with the AGS-16M8F bridging dose of 4.8 mg/kg given every 3 weeks.Results: The AGS-16M8F study (n = 26) closed before reaching the MTD. The median duration of treatment was 12 weeks (1.7-83 weeks). One subject had durable partial response (PR; 83 weeks) and 1 subject had prolonged stable disease (48 weeks). In the AGS-16C3F study (n = 34), the protocol-defined MTD was 3.6 mg/kg, but this was not tolerated in multiple doses. Reversible keratopathy was dose limiting and required multiple dose deescalations. The 1.8 mg/kg dose was determined to be safe and was associated with clinically relevant signs of antitumor response. Three of 13 subjects at 1.8 mg/kg had durable PRs (range, 100-143 weeks). Eight subjects at 2.7 mg/kg and 1.8 mg/kg had disease control >37 weeks (37.5-141 weeks).Conclusions: AGS-16C3F was tolerated and had durable antitumor activity at 1.8 mg/kg every 3 weeks. Clin Cancer Res; 24(18); 4399-406. ©2018 AACR.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models.

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

Role of efflux pumps and metabolising enzymes in drug delivery.

The impact of efflux pumps and metabolic enzymes on the therapeutic activity of various drugs has been well established. The presence of efflux pumps on various tissues and tumours has been shown to regulate the intracellular concentration needed to achieve therapeutic activity. The notable members of efflux proteins include P-glycoprotein, multi-drug resistance protein and breast cancer resistance protein. These efflux pumps play a pivotal role not only in extruding xenobiotics but also in maintaining the body's homeostasis by their ubiquitous presence and ability to coordinate among themselves. In this review, the role of efflux pumps in drug delivery and the importance of their tissue distribution is discussed in detail. To improve pharmacokinetic parameters of substrates, various strategies that modulate the activity of efflux proteins are also described. Drug metabolising enzymes mainly include the cytochrome P450 family of enzymes. Extensive drug metabolism due to the this family of enzymes is the leading cause of therapeutic inactivity. Therefore, the role of metabolising enzymes in drug delivery and disposition is extensively discussed in this review. The synergistic relationship between metabolising enzymes and efflux proteins is also described in detail. In summary, this review emphasises the urgent need to make changes in drug discovery and drug delivery as efflux pumps and metabolising enzymes play an important role in drug delivery and disposition.

In vivo antiviral efficacy of a dipeptide acyclovir prodrug, val-val-acyclovir, against HSV-1 epithelial and stromal keratitis in the rabbit eye model.

PURPOSE: A dipeptide prodrug of the antiviral nucleoside acyclovir (ACV), val-val-ACV (VVACV), was evaluated in vivo as a potential drug candidate for improving antiviral efficacy against herpetic epithelial and stromal keratitis. METHODS: The effect of 1% VVACV on epithelial keratitis induced by inoculation of HSV-1 strain McKrae (25 microL of 10(5) plaque-forming units [PFU]) in the scarified rabbit cornea and stromal keratitis induced by intrastromal injection of HSV-1 strain RE (10 microL of 10(5) PFU) was compared with that of 1% trifluorothymidine (TFT) and balanced salt solution as the vehicle control. Both eyes of 10 rabbits were used in each treatment group. Lesions were evaluated by slit lamp examinations over a 2-week period after infection. Aqueous humor samples and corneas were analyzed for drug concentrations at the end of each experiment. Cytotoxicity of VVACV in comparison with val-acyclovir (VACV), ACV, and TFT was evaluated in cellular proliferation assays. RESULTS: The dipeptide prodrug VVACV demonstrated excellent activity against HSV-1 in the rabbit epithelial and stromal keratitis models: 1% VVACV was as effective as 1% TFT. The prodrug was also less cytotoxic than TFT, which is the only effective drug currently licensed and routinely used for topical treatment of ocular herpes infections in the United States. CONCLUSIONS: The less cytotoxic and highly water-soluble prodrug VVACV, which showed excellent in vivo activity against HSV-1 in rabbit epithelial and stromal keratitis, is a promising drug candidate for treatment of ocular HSV infections.

Molecular evidence and functional expression of P-glycoprotein (MDR1) in human and rabbit cornea and corneal epithelial cell lines.

PURPOSE: Efflux pumps such as P-glycoprotein (P-gp; MDR1) are believed to be a major barrier to drug delivery. The purpose of this work was to determine whether cornea and corneal epithelial cells expresses the functionally active P-gp efflux pump. METHOD: Cultured rabbit primary corneal epithelial cells (rPCECs) and a corneal cell line (Statens Seruminstitut rabbit cornea [SIRC] cells) were selected as the model. Rhodamine-123 (Rho-123), a P-gp substrate, was used as a P-gp probe. To confirm gene expression, RT-PCR was performed with appropriate pairs of primers for rabbit and human MDR1. Subcloning, sequencing, and protein sequence determination were performed to confirm P-gp. RESULTS: Permeability of [(3)H] cyclosporin A (CsA) across SIRC cells was found at 1.74 x 10(-6) cm/s in the apical-to-basolateral and 5.1 x 10(-6) cm/s in the basolateral-to-apical directions. Uptake of Rho-123 across both SIRC cells and rPCECs was time and temperature dependent. Rho-123 uptake in SIRC cells was 14.4 picomoles/mg protein and in the presence of CsA (10 micro M) was 70.8 picomoles/mg protein at 3 hours. Uptake in rPCECs was the highest at 3 hours. Western blot analysis indicated a 170-kDa band confirming the presence of P-gp. Human cornea was also checked for the presence of P-gp. RT-PCR data indicated one single band, which was subcloned and sequenced to confirm the presence of P-gp. The protein sequence deduced from the fragment product indicated more than 89% homology with human MDR1. CONCLUSIONS: Functional and molecular characterization showed the existence of P-gp in human cornea, rabbit cornea, and a rabbit corneal cell line. This knowledge of the existence of P-gp will help in development of better ocular drug delivery strategies.

Current prodrug strategies via membrane transporters/receptors.

Prodrug design strategies have been employed to improve the delivery of drugs with undesirable pharmacokinetic properties such as chemical stability and lack of specificity. Targeted prodrug design represents a new strategy for site-directed and efficient drug delivery. Targeting of drugs to transporters and receptors to aid in site-specific carrier-mediated absorption is emerging as a novel and clinically significant approach. Various prodrugs have been successful in achieving the goals of enhanced bioavailability and are, therefore, considered to be an important tool in biopharmaceutics. This review highlights the advances in prodrug design targeted towards membrane transporters/receptors in the past few years.

Transporters/receptors in the anterior chamber: pathways to explore ocular drug delivery strategies.

Membrane transporters/receptors are involved in drug transport processes and play a key role in intestinal absorption, tissue distribution and elimination. Drug targeting to specific transporters and receptors using carrier-mediated absorption has immense clinical significance. Ocular drug delivery is a challenging task since it involves drug transport across various barriers in the eye. Specialised transport processes exist at these barriers, which control the entry of drugs and xenobiotics. Ocular drug therapy involving topical or systemic administration of drugs has various limitations. Transport processes in the eye have been targeted in an effort to increase ocular bioavailability of drugs following topical instillation. This review discusses various transport processes in the eye and drug delivery strategies utilising these transporters/receptors.

Effect of mono- and di-acylation on the ocular disposition of ganciclovir: physicochemical properties, ocular bioreversion, and antiviral activity of short chain ester prodrugs.

A series of short-chain carboxylic mono- and diesters of ganciclovir were synthesized in our laboratory. Physico-chemical properties, i.e., solubility (pH 4.2), partition coefficient in 1-octanol/phosphate buffer (pH 7.4), aqueous stability at various pH values, bioreversion kinetics in various ocular homogenates and effectiveness against various Herpes viruses in vitro were determined. The compounds exhibited a decrease in solubility as the ester length ascended with a corresponding increase in the octanol/buffer partition coefficient values. All of the prodrugs exhibit stability profiles typical of a carboxylic ester with maximum stability at neutral or slight acidic pH (4.0-7.0). Apparent first-order rate constants associated with prodrug to drug hydrolysis in the ocular homogenates varied depending on the size of the promoiety, lipophilicity of the compound, and the ocular tissue studied. The acetyl and butyryl mono and diesters were screened against various Herpes viruses. The monobutyrate ester of ganciclovir exhibits excellent activity against HSV-2 and VZV and provides a very high selectivity index against most of the viruses studied.

Validation of an ocular microdialysis technique in rabbits with permanently implanted vitreous probes: systemic and intravitreal pharmacokinetics of fluorescein.

The purpose of this work is to validate a novel ocular microdialysis sampling technique in rabbits with permanently implanted vitreous probes. This objective is achieved by studying the vitreous pharmacokinetics of fluorescein following systemic and intravitreal administration. The rabbits were divided into two groups (groups I and II) based on whether or not they were allowed a recovery period following surgical implantation of probes. The integrity of the blood-retinal barrier was determined by the vitreal protein concentrations and the fluorescein permeability index. Vitreal protein concentrations returned to baseline 48 h after probe implantation and therefore experiments were conducted 72 h post-implantation of probes in rabbits where recovery period was allowed. The permeability indices for fluorescein after systemic administration in group I (without recovery period) and group II (with recovery period) indicated that the integrity of the blood-retinal barrier was maintained and were found out to be 0.55 +/- 0.27 and 0.71 +/- 0.38%, respectively, for the vitreous chamber. Following microdialysis probe implantation in the group II rabbits, the blood-retinal barrier integrity was not compromised. A novel microdialysis technique in rabbits with permanently implanted probes for studying the pharmacokinetics of posterior segment has been developed and characterized.

Novel dipeptide prodrugs of acyclovir for ocular herpes infections: Bioreversion, antiviral activity and transport across rabbit cornea.

PURPOSE: A series of dipeptide prodrugs of antiviral nucleoside acyclovir (ACV) were designed to target the oligopeptide transporter on the cornea with an aim of improving the ocular bioavailability and therapeutic activity of ACV. METHODS: Aqueous stability, ocular bioreversion kinetics in various tissues, in vitro antiviral activity, cell proliferation assay and corneal transport characteristics of the dipeptide prodrugs were studied. Results. ACV dipeptide prodrugs were found to be more stable at pH 5.6 in comparison to L-Val-ACV, an amino acid prodrug of ACV. The prodrugs exhibited higher solubility than ACV. Val-Val-ACV and Val-Tyr-ACV were found to have excellent antiviral activity against herpes simplex virus-1 (HSV-1). All the dipeptide prodrugs exhibited lower cytotoxicity as compared to currently approved anti-HSV agent, trifluorothymidine (TFT). Transport of [(3)H] Val-ACV was inhibited significantly in the presence of the dipeptide prodrugs of ACV. Corneal permeabilities of all the ACV dipeptide prodrugs were observed to be higher than ACV possibly due to recognition of the prodrugs by the oligopeptide transporter on the cornea. CONCLUSIONS: The dipeptide prodrugs were found to be more permeable than the parent drug, ACV. More permeable, less cytotoxic ACV dipeptide prodrugs exhibited excellent chemical stability and antiviral activity against herpes simplex virus thereby rendering these lead compounds promising drug candidates against herpes virus infections.

Amino acid prodrugs of acyclovir as possible antiviral agents against ocular HSV-1 infections: interactions with the neutral and cationic amino acid transporter on the corneal epithelium.

PURPOSE: The aim of this study was to explore the feasibility of improvement of ocular bioavailability of the antiviral agent acyclovir by designing amino acid prodrugs targeted to the amino acid transporters on the rabbit cornea. MATERIALS AND METHODS: Transcorneal flux of two water-soluble amino acid ester prodrugs of acyclovir (ACV), gamma-glutamate-ACV (EACV) and L-tyrosine-ACV (YACV), was studied across freshly excised rabbit cornea. Chemical and enzymatic hydrolysis studies of the two prodrugs were also conducted. RESULTS: EACV inhibited the uptake of [(3)H]L-Arg in rabbit primary corneal epithelial cells (rPCECs). The compound also exhibited longer half-life (t(1/2)) in cornea in comparison to YACV. Transcorneal flux of EACV was observed to be concentration-, energy-, and sodium-dependent and independent of pH within the range studied. EACV transport was inhibited by neutral and cationic amino acids, L-ornithine (specific for cationic amino acids), and BCH (2-aminobicyclo-[2,2,1]-heptane-2-carboxylic-acid) (specific inhibitor for L-type system and B(0,+) system). On the other hand, YACV was not recognized by this amino acid transporter as it failed to inhibit the uptake of [(3)H]Arg, and also its transport across cornea was not inhibited by arginine. YACV and EACV exhibited excellent antiviral activity against HSV-1 and 2 and Varicella-Zoster Virus (VZV) in comparison to ACV. CONCLUSIONS: Analyses of the inhibition pattern of EACV transport suggests the involvement of a single transport system; namely, B(0,+). Design of amino acid prodrugs seems to be an attractive strategy to enhance the solubility of the otherwise poorly aqueous soluble compounds and also to afford a targeted and possibly enhanced delivery of the active drug.

In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates.

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters: Case studies.

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that "free" circulating antibodies are the pharmacologically active form needed to determine the PK/pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, "total" drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand/shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.

Developmental immunotoxicology assessment of rituximab in cynomolgus monkeys.

Rituximab is a chimeric murine/human-engineered immunoglobulin (Ig) G1 anti-CD20 monoclonal antibody, selectively depleting CD20-expressing cells in peripheral blood and lymphoid tissues. As part of the rituximab registration-enabling program for rheumatoid arthritis, cynomolgus monkey embryo-fetal development and pre- and postnatal developmental toxicity studies were performed. In both studies, female cynomolgus monkeys were administered rituximab iv at doses of 0/0, 15/20, 37.5/50, and 75/100 mg/kg (loading dose/study dose) from gestation day (GD) 20 to 50 for the embryo-fetal development study and GD 20 to postpartum (pp) day 28 for the pre- and postnatal study. In the embryo-fetal development study, although maternal dosing ended during the first trimester at GD 50, placental transfer of rituximab to fetuses was demonstrated at GD 100. Consequently, fetuses demonstrated B-cell depletion in lymphoid tissues at GD 100. Repletion of B cells was demonstrated in infants in a follow-up pre- and postnatal study following fetal and neonatal exposure. In the pre- and postnatal study, despite B-cell depletion, there was no significant functional consequence on the infant's ability to mount T-cell-dependent antibody responses following vaccination or antigenic challenge. Overall, rituximab was well tolerated at maximum feasible doses up to 100 mg/kg in pregnant cynomolgus monkeys and their infants after exposure from the period of organogenesis throughout pregnancy, parturition, and postnatal development. Importantly, the preclinical data have been concordant with the clinical data in children for cases where rituximab was administered during pregnancy.

In vivo ocular pharmacokinetics of acyclovir dipeptide ester prodrugs by microdialysis in rabbits.

In vivo corneal absorption of the dipeptide prodrugs of acyclovir (ACV) was evaluated using microdialysis in rabbits. A corneal well was placed on the cornea of the anesthetized New Zealand White rabbits with implanted linear probes into the aqueous humor. Two hundred microliters of a 1% solution of L-valine-ACV (VACV), glycine-valine-ACV (GVACV), valine-valine-ACV (VVACV), and valine-tyrosine-ACV (VYACV) was placed in the corneal well and was allowed to diffuse for a period of 2 h, following which the drug solution was aspirated and well removed. Samples were collected every 20 min throughout the infusion and postinfusion phases and were analyzed by HPLC to obtain the aqueous humor concentrations. Absorption rate constants of all the compounds were found to be lower than the elimination rate constants. GVACV exhibited highest absorption rate (ka) compared with other prodrugs, but all the prodrugs showed similar terminal elimination rate (lambda(z)). The time of maximum absorption (Tmax) of ACV after administration of VACV and the dipeptide prodrugs did not vary significantly (p < 0.05). GVACV exhibited the highest concentration (Cmax) and area under curve (AUC) upon absorption (p < 0.05) compared to VACV, VVACV, and VYACV. Dipeptide prodrugs of ACV were absorbed through the cornea at similar rates but to varying extents. The dipeptide prodrug GVACV owing to its enhanced absorption of ACV seems to be a promising candidate for the treatment of ocular HSV infections.

Pharmacokinetics/pharmacodynamics of nondepleting anti-CD4 monoclonal antibody (TRX1) in healthy human volunteers.

PURPOSE: TRX1 is a nondepleting anti-CD4 monoclonal IgG1 antibody being developed to induce tolerance by blocking CD4-mediated functions. The purpose of this study is to describe the pharmacokinetics (PK) and pharmacodynamics (PD) of TRX1 and to develop a receptor-mediated PK/PD model that characterizes the relationships between serum TRX1 concentration and total and free CD4 expression in healthy male volunteers. METHODS: Nine subjects from three dosing cohorts in double-blinded, placebo-controlled phase I clinical study was included in the analysis. Serum TRX1 levels were determined using enzyme-linked immunosorbent assay. Blood total and free CD4 receptor levels were determined by using flow cytometric analyses. The receptor-mediated PK/PD model was developed to describe the dynamic interaction of TRX1 binding with CD4 receptors. RESULTS AND CONCLUSIONS: TRX1 displayed nonlinear pharmacokinetic behavior and the CD4 receptors on T cells were saturated and down-modulated following treatment with TRX1. Results from in vitro studies using purified human T cells suggested that CD4-mediated internalization may constitute one pathway by which CD4 is down-modulated and TRX1 is cleared in vivo. The developed receptor-mediated PK/PD model adequately described the data. This PK/PD model was used to simulate PK/PD time profiles after different dosing regimens to help guide the dose selection in future clinical studies.