Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

Container Closure and Delivery Considerations for Intravitreal Drug Administration.

Intravitreal (IVT) administration of therapeutics is the standard of care for treatment of back-of-eye disorders. Although a common procedure performed by retinal specialists, IVT administration is associated with unique challenges related to drug product, device and the procedure, which may result in adverse events. Container closure configuration plays a crucial role in maintaining product stability, safety, and efficacy for the intended shelf-life. Careful design of primary container configuration is also important to accurately deliver small volumes (10-100 µL). Over- or under-dosing may lead to undesired adverse events or lack of efficacy resulting in unpredictable and variable clinical responses. IVT drug products have been traditionally presented in glass vials. However, pre-filled syringes offer a more convenient administration option by reducing the number of steps required for dose preparation there by potentially reducing the time demand on the healthcare providers. In addition to primary container selection, product development studies should focus on, among other things, primary container component characterization, material compatibility with the formulation, formulation stability, fill volume determination, extractables/leachables, and terminal sterilization. Ancillary components such as disposable syringes and needles must be carefully selected, and a detailed administration procedure that includes dosing instructions is required to ensure successful administration of the product. Despite significant efforts in improving the drug product and administration procedures, ocular safety concerns such as endophthalmitis, increased intraocular pressure, and presence of silicone floaters have been reported. A systematic review of available literature on container closure and devices for IVT administration can help guide successful product development.

Intracellular delivery of the reactive oxygen species generating agent D-penicillamine upon conjugation to poly-L-glutamic acid.

D-penicillamine is an aminothiol that is cytotoxic to cancer cells and generates dose dependent reactive oxygen species (ROS) via copper catalyzed oxidation. However, the delivery of D-pen to cancer cells remains a challenge due to its high hydrophilicity, highly reactive thiol group and impermeability to the cell membrane. To overcome this challenge, we investigated a novel poly-L-glutamic acid (PGA) conjugate of D-pen (PGA-D-pen) where D-pen was conjugated to PGA modified with 2-(2-pyridyldithio)-ethylamine (PDE) via disulfide bonds. Confocal microscopy and cell uptake studies showed that the fluorescently labeled PGA-D-pen was taken up by human leukemia cells (HL-60) in a time dependent manner. Treatment of HL-60, murine leukemia cells (P388) and human breast cancer cells (MDA-MB-468) with PGA-D-pen resulted in dose dependent cytotoxicity and elevation of intracellular ROS levels. PGA-D-pen induced apoptosis in HL-60 cells which was verified by Annexin V binding. The in vivo evaluation of the conjugate in the P388 murine leukemia model (intraperitoneal) resulted in significant enhancement in the survival of CD2F1 mice over vehicle control.

Enhanced intracellular delivery of the reactive oxygen species (ROS)-generating copper chelator D-penicillamine via a novel gelatin--D-penicillamine conjugate.

D-Penicillamine (D-pen) is an established copper chelator. We have recently shown that the copper-catalyzed D-pen oxidation generates concentration-dependent hydrogen peroxide (H 2O 2). Additionally, D-pen coincubated with cupric sulfate resulted in cytotoxicity in human leukemia and breast cancer cells due to the extracellular generation of reactive oxygen species (ROS). The inherent physicochemical properties of D-pen such as its short in vivo half-life, low partition coefficient, and rapid metal catalyzed oxidation limit its intracellular uptake and the potential utility as an anticancer agent in vivo. Therefore, to enhance the intracellular delivery and to protect the thiol moiety of D-pen, we designed, synthesized, and evaluated a novel gelatin-D-pen conjugate. D-pen was covalently coupled to gelatin with a biologically reversible disulfide bond with the aid of a heterobifunctional cross-linker ( N-succinimidyl-3-(2-pyridyldithio)-propionate) (SPDP). Additionally, fluorescein-labeled gelatin-D-pen conjugate was synthesized for cell uptake studies. D-pen alone was shown not to enter leukemia cells. In contrast, the qualitative intracellular uptake of the conjugate in human leukemia cells (HL-60) was shown with confocal microscopy. The conjugate exhibited slow cell uptake (over the period of 48 to 72 h). A novel HPLC assay was developed to simultaneously quantify both D-pen and glutathione in a single run. The conjugate was shown to completely release D-pen in the presence of glutathione (1 mM) in approximately 3 h in PBS buffer, pH 7.4. The gelatin-D-pen conjugate resulted in significantly greater cytotoxicity compared to free D-pen, gelatin alone, and a physical mixture of gelatin and D-pen in human leukemia cells. Further studies are warranted to assess the potential of D-pen conjugate in the delivery of D-pen as a ROS generating anticancer agent.

Cross-linked guar gum hydrogel discs for colon-specific delivery of ibuprofen: formulation and in vitro evaluation.

Hydrogel discs of guar gum cross-linked with glutaraldehyde were prepared as vehicles for colon-specific drug delivery. Ibuprofen was chosen as model drug. The discs were evaluated for such parameters as size, shape, weight, and drug loading. Swelling (buffer uptake) and in vitro drug release study, in presence and absence of rat caecal contents, was performed in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4) to evaluate the effect of various formulation parameters like guar gum concentration, amount of cross-linking agent, and cross-linking time on drug release. Cross-linking resulted in significant reduction in swelling of guar gum. Significant increase in drug release was observed in medium containing rat caecal content. Percent drug release increased with increasing glutaraldehyde concentration. Cross-linking time and guar gum concentration did not have any significant effect on drug release in the range studied.

Floating microspheres of cimetidine: formulation, characterization and in vitro evaluation.

The present study involves preparation and evaluation of floating microspheres with cimetidine as model drug for prolongation of gastric residence time. The microspheres were prepared by the solvent evaporation method using polymers hydroxypropylmethyl cellulose and ethyl cellulose. The shape and surface morphology of prepared microspheres were characterized by optical and scanning electron microscopy, respectively. In vitro drug release studies were performed and drug release kinetics was evaluated using the linear regression method. Effects of the stirring rate during preparation, polymer concentration, solvent composition and dissolution medium on the size of microspheres and drug release were also observed. The prepared microspheres exhibited prolonged drug release (approximately 8 h) and remained buoyant for > 10 h. The mean particle size increased and the drug release rate decreased at higher polymer concentration. No significant effect of the stirring rate during preparation on drug release was observed. In vitro studies demonstrated diffusion-controlled drug release from the microspheres.

Polymer-Drug Conjugates for Anticancer Drug Delivery.

Polymer-drug conjugates (PDCs) are drug delivery systems where one or more drug(s) are covalently attached to the functional groups of the polymer directly or through a spacer. Several anticancer drugs that have been used to synthesize PDCs are currently under clinical trials. PDCs have shown enhanced tumor accumulation, increased therapeutic index, and prolonged circulation, accompanied by a sustained release of the bound drug. Distinct cell uptake mechanisms make PDCs less sensitive to efflux pumps associated with the development of multi-drug resistance. However, the effectiveness of PDCs as a delivery system primarily depends on the drug, polymer, type of linkage, and presence of targeting groups. Due to the availability of different functional groups and spacers, it is possible to control drug release as well as multi-functionalize PDCs, thereby increasing their versatility as drug carriers. Furthermore, active tumor uptake may be achieved by using the concept of drug targeting. However, functionalization alters the in vivo behavior of the polymer, signifying the evaluation of safety and effectiveness of PDCs. Several PDCs are currently being tested in different phases of clinical trials. This review focuses on critical aspects in the design of PDCs when used in cancer drug delivery.

D-penicillamine and other low molecular weight thiols: review of anticancer effects and related mechanisms.

Low molecular weight thiols (LMWTs) like N-acetyl cysteine, D-penicillamine, captopril, Disulfiram and Amifostine, etc. have been used as chemo-preventive agents. Recent studies have reported cell growth inhibition and cytotoxicity in several different types of cancer cells following treatment with several LMWTs. Cytotoxic and cytostatic effects of LMWTs may involve interaction of the thiol group with cellular lipids, proteins, intermediates or enzymes. Some of the mechanisms that have been proposed include a p53 mediated apoptosis, thiyl radical induced DNA damage, membrane damage through lipid peroxidation, anti-angiogenic effects induced by inhibition of matrix metalloproteinase enzymes and angiostatin generation. LMWTs are strong chelators of transition metals like copper, nickel, zinc, iron and cobalt and may cause metal co-factor depletion resulting in cytotoxicity. Oxidation of thiol group can also generate cytotoxic reactive oxygen species (ROS).

Polypeptide conjugates of D-penicillamine and idarubicin for anticancer therapy.

We investigated anticancer therapy with a novel combination of D-penicillamine (D-pen) and Idarubicin (Ida) in a synthetic dual drug conjugate (DDC). D-pen and Ida were covalently linked to poly(α)-L-glutamic acid (PGA) via reducible disulfide and acid-sensitive hydrazone bonds, respectively. The DDCs showed cell uptake and sustained release of the bound drugs in conditions mimicking the intracellular release media (10mM glutathione and pH 5.2). The in-vitro cytotoxicity of DDCs was comparable to unconjugated Ida in several sensitive and resistant cancer cell lines and correlated with the rate of cell uptake. In a single equivalent-dose pharmacokinetic study, DDCs enhanced the drug exposure by 7-fold and prolonged the plasma circulation half-life (t(1/2)) by 5-fold over unconjugated Ida. The therapeutic index of DDCs was 2-3-fold higher than unconjugated drugs. DDCs caused 89% tumor growth inhibition compared to 60% by unconjugated Ida alone and led to significant enhancement in the median survival (17%) of athymic nu/nu mice bearing NCI-H460 tumor xenografts.

Lipid nanocapsule as vaccine carriers for his-tagged proteins: evaluation of antigen-specific immune responses to HIV I His-Gag p41 and systemic inflammatory responses.

The purpose of this study was to design novel nanocapsules (NCs) with surface-chelated nickel (Ni-NCs) as a vaccine delivery system for histidine (His)-tagged protein antigens. Ni-NCs were characterized for binding His-tagged model proteins through high-affinity non-covalent interactions. The mean diameter and zeta potential of the optimized Ni-NCs were 214.9 nm and -14.8 mV, respectively. The optimal binding ratio of His-tagged Green Fluorescent Protein (His-GFP) and His-tagged HIV-1 Gag p41 (His-Gag p41) to the Ni-NCs was 1:221 and 1:480 w/w, respectively. Treatment of DC2.4 cells with Ni-NCs did not result in significant loss in the cell viability up to 24h (<5%). We further evaluated the antibody response of the Ni-NCs using His-Gag p41 as a model antigen. Formulations were administered subcutaneously to BALB/c mice at day 0 (prime) and 14 (boost) followed by serum collection on day 28. Serum His-Gag p41-specific antibody levels were found to be significantly higher at 1 and 0.5 µg doses of Gag p41-His-Ni-NCs (His-Gag p41 equivalent) compared with His-Gag p41 (1 µg) adjuvanted with aluminum hydroxide (AH). The serum IgG2a levels induced by Gag p41-His-Ni-NCs (1 µg) were significantly higher than AH adjuvanted His-Gag p41. The Ni-NCs alone did not result in the elevation of systemic IL-12/p40 and CCL5/RANTES inflammatory cytokine levels upon subcutaneous administration in BALB/c mice. In conclusion, the proposed Ni-NCs can bind His-tagged proteins and have the potential to be used as antigen delivery system capable of generating strong antigen-specific antibodies at doses much lower than with aluminum-based adjuvant and causing no significant elevation of systemic pro-inflammatory IL-12/p40 and CCL5/RANTES cytokines.

In vitro and in vivo assessment of targeting lipid-based nanoparticles to the epidermal growth factor-receptor (EGFR) using a novel Heptameric ZEGFR domain.

Lipid-based oil-filled nanoparticles (NPs) with a high concentration of surface-chelated nickel (Ni-NPs) were successfully prepared using a Brij 78-NTA-Ni conjugate synthesized with Brij 78 (Polyoxyethylene (20) stearyl ether) and nitrilotriacetic acid (NTA). The facile incorporation of the Brij 78-NTA-Ni conjugate into the NP formulation allowed up to 90% Ni incorporation, which was a significant improvement over the previously used standard agent DOGS-NTA-Ni which led to ~6% Ni incorporation. The Ni-NPs were targeted to the highly epidermal growth factor receptor (EGFR)-overexpressing epidermoid carcinoma cells A431. This was accomplished using a novel high affinity histidine×6-tagged EGFR-binding Z domain (heptameric Z(EGFR) domain). In vitro cell uptake studies showed enhanced internalization (up to 90%) of the targeted Ni-NPs in A431 cells with only ≤10% internalization of the untargeted Ni-NPs. ICP-MS analysis used to quantify the amount of Ni in the cells were in close agreement with flow cytometry studies, which showed a dose dependent increase in the amount of Ni with the targeted Ni-NPs. Cell uptake competition studies showed that internalization of the targeted Ni-NPs within the cells was competed off with free heptameric Z(EGFR) domain at concentrations of 8.75ng/mL or higher. In vivo studies were carried out in nude mice bearing A431 tumors to determine the biodistribution and intracellular delivery. Near Infrared (NIR) optical imaging studies using Alexa750-labeled heptameric Z(EGFR) domain showed localization of 19% of the total detected fluorescence intensity in the tumor tissue, 28% in the liver and 42% in the kidneys 16h post i.v. injection. ICP-MS analysis showed almost a two-fold increase in the amount of intracellular Ni with the targeted Ni-NPs. These new Ni-NPs could be a very useful tool for targeting and drug delivery to a wide range of EGFR positive cancers.

Oral sustained delivery of atenolol from floating matrix tablets-formulation and in vitro evaluation.

Floating matrix tablets of atenolol were developed to prolong gastric residence time and increase drug bioavailability. Atenolol was chosen as a model drug because it is poorly absorbed from the lower gastrointestinal tract. The tablets were prepared by direct compression technique, using polymers such as hydroxypropyl methylcellulose (HPMC K15M, K4M), guargum (GG), and sodium carboxymethylcellulose (SCMC), alone or in combination, and other standard excipients. Tablets were evaluated for physical characteristics viz. hardness, swelling index, floating capacity, thickness, and weight variation. Further, tablets were evaluated for in vitro release characteristics for 8 hr. The effect of effervescent on buoyancy and drug release pattern was also studied. In vitro release mechanism was evaluated by linear regression analysis. GG- and SCMC-based matrix tablets showed significantly greater swelling indices compared with other batches. The tablets exhibited controlled and prolonged drug release profiles while floating over the dissolution medium.