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.

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.

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 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.

Mechanism of corneal permeation of L-valyl ester of acyclovir: targeting the oligopeptide transporter on the rabbit cornea.

PURPOSE: To delineate mechanisms associated with the corneal transport of a L-valine prodrug of an antiviral agent, acyclovir. METHOD: The permeability and enzymatic hydrolysis of L-Val-ACV were evaluated using freshly excised rabbit cornea. Transport mechanism across rabbit cornea was investigated through a competitive inhibition study of L-Val-ACV with other substrates of human peptide transporter (hPepT1). RESULTS: L-Valyl ester of Acyclovir (L-Val-ACV) was approximately threefold more permeable across the intact rabbit cornea than acyclovir (ACV). Dipeptides, beta-lactam antibiotics, and angiotensin converting enzyme (ACE) inhibitors, strongly inhibited the transport of L-Val-ACV indicating that a carrier mediated transport system specific for peptides is primarily responsible for the corneal permeation of L-Val-ACV. L-Val-ACV transport was found to be saturable (Km = 2.26 +/- 0.34 mM, Jmax = 1.087 +/- 0.05 nmoles cm(-2) min(-1)), energy and pH dependent. CONCLUSIONS Functional evidence of an oligopeptide transport system present on the rabbit cornea has been established. The peptide transporter on the corneal epithelium may be targeted to improve the ocular bioavailability of poorly absorbed drugs.

Pharmacokinetics of novel dipeptide ester prodrugs of acyclovir after oral administration: intestinal absorption and liver metabolism.

The amino acid prodrug of acyclovir (ACV), valacyclovir (VACV), is an effective antiherpetic drug. Systemic availability of ACV in humans is 3 to 5 times higher after oral administration of VACV. Enhanced bioavailability of VACV has been attributed to its carrier-mediated intestinal absorption via hPEPT1 peptide transporter followed by rapid and complete conversion to ACV. An earlier report suggested that the dipeptide ester prodrugs of ACV possess high affinity toward the intestinal oligopeptide transporter hPEPT1 and therefore seem to be promising candidates in the treatment of oral herpes virus infections. In the present study, we have examined the bioavailability of a series of dipeptide prodrugs of ACV after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The area under plasma-concentration time curves expressed as minutes microgram milliliter(-1) for total concentration of VACV (208.4 +/- 41.2), and the dipeptide prodrugs Gly-Val-ACV (GVACV) (416.1 +/- 140.9), Val-Val-ACV (VVACV) (147.7 +/- 89.3), and Val-Tyr-ACV (VYACV) (180.7 +/- 81.2) were significantly higher than that of ACV (21.2 +/- 5.2) upon intestinal absorption. Interestingly, the bioavailability of ACV after administration of GVACV was approximately 2-fold higher than VACV. There was significant metabolism by hepatic first pass effect of the dipeptide prodrugs as evident by the higher levels of ACV obtained after systemic absorption compared with intestinal absorption of GVACV and VVACV. The dipeptide prodrugs of ACV exhibited higher systemic availability of regenerated ACV upon oral administration and thus seem to be promising drug candidates in treatment of genital herpes infections.

Interactions of the dipeptide ester prodrugs of acyclovir with the intestinal oligopeptide transporter: competitive inhibition of glycylsarcosine transport in human intestinal cell line-Caco-2.

The oligopeptide transporter may be exploited to enhance the absorption of drugs by synthesizing their dipeptide ester prodrugs, which may be recognized as its substrates. Various dipeptide esters of acyclovir (ACV), an antiviral nucleoside analog, were synthesized. Enzymatic hydrolysis and affinity of the prodrugs toward the human intestinal peptide transporter hPEPT1 were studied using the human intestinal Caco-2 cell line. Affinity studies were performed by inhibiting the uptake of [(3)H]glycylsarcosine by the prodrugs. The uptake of glycylsarcosine was found to be saturable at higher concentrations and was competitively inhibited by the prodrugs of ACV. All prodrugs except Tyr-Gly-ACV demonstrated a higher affinity (1.41-4.96 mM) toward hPEPT1 than cephalexin (8.19 +/- 2.12 mM), which was used as a positive control. Two prodrugs, Gly-Val-ACV and Val-Val-ACV, showed comparable affinity to Val-ACV, an amino acid prodrug of ACV recognized by PEPT1/PEPT2. The permeability of Gly-Val-ACV (2.99 +/- 0.59 x 10(-6) cm/s) across Caco-2 was comparable with that of Val-ACV (3.01 +/- 0.21 x 10(-6) cm/s) and was significantly inhibited (63%) in presence of glycylsarcosine. The transport of GVACV across Caco-2 was saturable at higher concentrations, and the parameters were calculated as K(m) 3.16 +/- 0.31 mM and V(max) 0.014 +/- 0.00058 nmol cm(-2) min(-1). Overall, the results suggest that the dipeptide prodrugs of ACV have a high affinity toward the intestinal oligopeptide transporter hPEPT1 and therefore seem to be promising candidates in the treatment of ocular and oral herpesvirus infections, because cornea and intestinal epithelia seem to express the oligopeptide transporters.

Mechanism of a model dipeptide transport across blood-ocular barriers following systemic administration.

The purposes of this study were to provide functional evidence for the presence of a peptide transporter on blood-ocular barriers and to elucidate the mechanism of a dipeptide transport across these barriers following systemic administration. Glycylsarcosine was chosen as a model dipeptide and [(3)H] glycylsarcosine was administered through the marginal ear vein of New Zealand white rabbits. At the end of an experimental period, vitreous humor, retina and aqueous humor were collected. Time dependent uptake of glycylsarcosine into ocular tissues was studied at 5, 10, 15 and 30 min. Competitive inhibition studies were performed by intravenous administration of [(3)H] glycylsarcosine with and without various inhibitors. Concentration-dependent ocular uptake of glycylsarcosine was carried out by administration of various concentrations of unlabelled glycylsarcosine spiked with a fixed amount of [(3)H] glycylsarcosine. Time-dependent uptake of glycylsarcosine into vitreous humor, retina and aqueous humor for a period of 30 min following systemic administration was linear. Ocular uptake of glycylsarcosine was inhibited by peptide transporter substrates such as dipeptides (glycylproline and carnosine) and captopril but not by non-substrates such as amino acids. Concentration-dependent self-inhibition of glycylsarcosine ocular uptake was also observed. The results indicate that model dipeptide is transported across blood-ocular barriers via a carrier-mediated process. In conclusion, an oligopeptide transport system is involved in the transport of glycylsarcosine across blood-ocular barriers. This information may be utilized to design transporter/receptor targeted drug delivery systems for efficient ocular uptake from systemic administration.