Polyoxazoline polymers with methyl (PMOZ), ethyl (PEOZ), and propyl (PPOZ) side chains were prepared by the living cationic polymerization method and purified by ion-exchange chromatography. The ...following properties of polyoxazoline (POZ) were measured: apparent hydrodynamic radius by aqueous size-exclusion chromatography, relative lipophilicity by reverse-phase chromatography, and viscosity by cone–plate viscometry. The PEOZ polymers of different molecular weights were first functionalized and then conjugated to model biomolecules such as bovine serum albumin, catalase, ribonuclease, uricase, and insulin. The conjugates of catalase, uricase, and ribonuclease were tested for in vitro activity using substrate-specific reaction methods. The conjugates of insulin were tested for glucose lowering activity by injection to naïve Sprague–Dawley rats. The conjugates of BSA were injected into New Zealand white rabbits and serum samples were collected periodically and tested for antibodies to BSA. The safety of POZ was also determined by acute and chronic dosing to rats. The results showed that linear polymers of POZ with molecular weights of 1 to 40 kDa can easily be made with polydispersity values below 1.10. Chromatography results showed that PMOZ and PEOZ have a hydrodynamic volume slightly lower than PEG; PEOZ is more lipophilic than PMOZ and PEG; and PEOZ is significantly less viscous than PEG especially at the higher molecular weights. When PEOZ was attached to the enzymes catalase, ribonuclease, and uricase, the in vitro activity of the resultant bioconjugates depended on the extent of protein modification. POZ conjugates of insulin lowered blood glucose levels for a period of 8 h when compared to 2 h for insulin alone. PEOZ, like PEG, was also able to successfully attenuate the immunogenic properties of BSA. The POZ polymers (10 and 20 kDa) are safe when administered intravenously to rats, and the maximum tolerated dose (MTD) was greater than 2 g/kg. Blood counts, serum chemistry, organ weights, and the histopathology of key organs were normal. These results conclude that POZ has the desired drug delivery properties for a new biopolymer.
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The potential of poly (2-oxazoline) or POZ to be a versatile and broad based platform for drug delivery with wide utility in multiple therapeutic areas has long been recognized by ...experts in the field. This feature article provides a case study which describes the chemistry and preclinical studies underlying the Investigational New Drug Application for SER-214, a POZ conjugate of rotigotine, for the treatment of Parkinson’s disease. We report the chemistry, preclinical safety and pharmacology, and the early clinical safety, tolerability and pharmacokinetic data from the Phase I study in patients. SER-214 utilizes a POZ polymer and proprietary custom linker technology to deliver a sustained dose of rotigotine over a period of seven days following a single subcutaneous administration – a result not observed by any other polymer approach that we are aware of. As such, this candidate drug has the promise to be a major advancement in the treatment of Parkinson’s disease. Furthermore, this feature article also highlights the versatility of the POZ polymer platform (POZ™) to deliver cancer drugs by actively targeting cancer cells. Preclinical data reported in this feature showcase the polymer’s attributes in facilitating targeted delivery with folic acid and antibody targeting agents (ADCs). The ability of POZ to reliably delivery large payloads of anticancer drugs is of particular importance when pursuing low-receptor-density targets on the cancer cell. The data presented in this feature, much of it for the first time, establish the broad utility of the POZ polymer platform in drug development. Together with its ease of manufacture, ability to attach drugs to the polymer, and ability to administer an appropriate dose to patients, the results underscore the need to further explore and expand the untapped potential of POZ for the development of new therapeutics for unmet medical needs.
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Poly(2-oxazoline)-drug conjugates with drugs attached via releasable linkages are being developed for drug delivery. Such conjugates with pendent ester linkages that covalently bind ...drugs to the polymer backbone exhibit significantly slower hydrolytic release rates in plasma than the corresponding PEG- and dextran-drug conjugates. The slow drug release rates in-vitro of these POZ-drug conjugates contribute to extended in-vivo pharmacokinetic profiles. In some instances, the release kinetics may be relatively sustained and ideal for once-a-week subcutaneous injection, whereas the native drug by itself may only have an in-vivo half-life of a few hours. The origin of this unusual kinetic and pharmacokinetic behavior is proposed here to involve folding of the POZ conjugate such that the relatively hydrophobic drug forms a central core, and the relatively hydrophilic polymer wraps around the core and slows enzymatic attack on the drug-polymer chemical linkage. Here we present evidence supporting this hypothesis and demonstrate how the hypothesis can be used to tune hydrolytic release rates and pharmacokinetics. Evidence for the folding hypothesis is taken from hydrolysis kinetics of a range of drugs in plasma, pharmacokinetics of a range of drugs following subcutaneous injection in laboratory animals, and nuclear magnetic resonance (NMR) studies showing folding of the POZ-rotigotine molecule. The drugs included in this study to test the hypothesis are: rotigotine, buprenorphine, dexanabinol, cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC) and cannabigerol (CBG).
Poly(2-ethyl 2-oxazoline) (PEOZ) is a water-soluble, stable and biocompatible polymer that was prepared in a linear form for the conjugation of protein biomolecules. Polymers of molecular weights ...ranging from 5 to 20kDa, with an aldehyde or an amine functional terminal group, were synthesized with narrow polydispersities. To assess the suitability of the polymer for therapeutic application, granulocyte colony stimulating factor (G-CSF) was used as a model protein for PEOZ conjugation. Two coupling strategies were employed, namely the chemical N-terminal reductive amination and the enzymatic transglutaminase (TGase) mediated glutamine conjugation. The secondary structure of the protein, measured by circular dichroism, was maintained upon PEOZylation and the stability of conjugates toward aggregation at 37°C was improved compared to G-CSF. The potency of PEOZ-G-CSF mono-conjugates was tested in vitro by cell proliferation assays and in vivo by studying the effects on white blood cell and neutrophil count increases in normal rats. The results have shown that PEOZ is suitable for protein conjugation by both chemical and enzymatic methods and that the conjugates of G-CSF retained high biological activity, both in vitro and in vivo.
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ABSTRACT
Currently available dopaminergic drugs such as levodopa and dopamine (DA) receptor agonists impart considerable improvement in Parkinson's disease (PD) motor symptoms but often lead to ...significant motor complications including “wearing‐off” and dyskinesia. Such complications are believed to stem from the pulsatile nature of dopaminergic stimulation with these agents. Continuous dopaminergic drug delivery using polyoxazoline (POZ) polymer conjugation may improve motor symptoms, while avoiding development of side effects. The purposes of the current study are to characterize the in vitro and in vivo pharmacokinetics of POZ conjugation of a U.S. Food and Drug Administration (FDA)‐approved DA agonist, rotigotine, and to evaluate their effects in an established rat model of PD. After determination of release profiles of several POZ‐conjugated constructs (“fast”: SER‐212; “moderate”: SER‐213; and “slow”: SER‐214) using in vitro hydrolysis, normal male Sprague‐Dawley rats were used for determination of the pharmacokinetic profile of both acute and chronic exposure. Finally, a separate group of rats was rendered hemiparkinsonian using intracranial 6‐hydroxydopamine (6‐OHDA) infusions, treated acutely with POZ‐rotigotine, and assessed for rotational behavior and antiparkinsonian benefit using the cylinder test. POZ‐rotigotine formulations SER‐213 and SER‐214 led to substantial pharmacokinetic improvement compared to unconjugated rotigotine. In addition, SER‐214 led to antiparkinsonian effects in DA‐lesioned rats that persisted up to 5 days posttreatment. Repeated weekly dose administration of SER‐214 to normal rats for up to 12 weeks demonstrated highly reproducible pharmacokinetic profiles. The continuous dopaminergic stimulation profile afforded by SER‐214 could represent a significant advance in the treatment of PD, with potential to be a viable, once‐per‐week therapy for PD patients.
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The pharmacokinetic parameters of peldesine (BCX‐34) were investigated after single and multiple oral doses in two groups of healthy adult volunteers. The pharmacodynamic elevation of endogenous ...inosine and 2′‐deoxyguanosine was simultaneously monitored. The first group of 8 subjects received an intravenous dose (18 mg/m2) and five oral doses (30, 63, 108, 144, and 192 mg/m2) of drug. A second group of 12 subjects received 160 mg/m2 in four and in six divided doses orally Serial blood samples and total urine outputs were collected during dosing and for at least 24 hours after the last dose was administered. One set of samples was analyzed using high‐pressure liquid chromatography/ultraviolet (LC/UV) methods, validated for intact drug in human plasma and urine samples. Another set of samples was analyzed for the biomarkers, inosine and 2′‐deoxyguanosine, using high‐pressure LC with either mass spectrometry (MS) or electrochemical detection (EC) methods. The pharmacokinetic parameters of inosine and 2′‐deoxyguanosine were calculated using noncompartmental methods and correlated against the pharmacokinetic parameters of BCX‐34. For the single‐dose study, the results exhibited linear pharmacokinetics over the dose range from 30 to 144 mg/m2. The calculated terminal half‐life was 3.5 ± 1.0 h, and the absolute bioavailability of the oral formulation was approximately 51%. Analysis of urine in the first 24 hours of collection accounted for approximately 82% of the absorbed intact drug. Evaluation of the multiple‐dose pharmacokinetics indicated that steady‐state blood concentrations were achieved by 24 hours when the drug was administered four or six times a day. A drug dose‐related elevation of plasma 2′‐deoxyguanosine was observed. This phenomenon was not seen with plasma inosine levels. However, analysis of urine samples showed an increase in inosine output with an increase in the drug dose. The calculated terminal half‐life of inosine and 2′‐deoxyguanosine was 15.3 ± 1.8 h and 1.3 ± 0.1 h, respectively.
In-vitro studies were conducted to study the efficacy of mixed and self-emulsifying creams and hydrophobic ointment formulations in delivering peldesine (BCX-34) into and across cryopreserved human ...cadaver skin (HCS). Oil-in-water cream formulations, containing 1% w/w of radiolabeled C
14 BCX-34 and propylene glycol (PG), glycerin (GLY), isopropyl myristate (IPM), oleic acid (OA) and capric-caprylic esters (CE) were prepared. Petrolatum and lanolin based ointments were also prepared with PG. Sections of the HCS, 250 μm thick, were fitted to vertical Franz diffusion chambers containing a receptor medium of pH 7.4 phosphate buffer solution maintained at 37°C. Using the finite dose technique, 4–6 mg of a formulation sample was applied to the epidermal surface of each section and drug diffusion was permitted for 12 and 24 h periods. The distribution of drug into the HCS epidermis, dermis and into the receptor medium was measured by scintillation spectroscopy. The results show good correlation of the calculated in-vitro values for flux and skin-vehicle partition coefficients against the observed amounts of drug detected in the HCS. The mixed emulsion cream formulation containing PG delivered higher amounts of drug into the skin when compared to the same formulation containing GLY cream. The self-emulsifying cream formulation containing IPM had a higher skin-vehicle partition coefficient and delivered more drug into the dermis when compared to those formulations that contained OA and CE. The petrolatum ointment delivered six times more drug into the epidermis than the lanolin ointment, and had higher skin-vehicle partition values. In conclusion, creams containing PG and petrolatum-base formulations would be suitable for BCX-34 dermal delivery.
Solutions of non-ionic surfactants were prepared in water and buffer. The osmotic behavior of these solutions were studied by measuring their osmolality, using the freezing point depression method. ...Graphical representation of osmolality versus concentration depicted non-linear parabolic curves that were applied to logarithmic and polynomial mathematical models. One such surfactant, poloxamer 407, was selected and studied in solutions of water and buffer. We observed curves of similar profile, which were attributed to its interaction and association in aqueous solution. In the liquid state the osmotic influence is due to the surfactant-water interaction. When the solution is warmed and converted to the gel state, the polymer is taken out of play and the osmotic influence is due to the aqueous solution only. This phenomenon was supported by in vitro red blood cell tonicity observations.
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