Charles D. Conover

Effective drug delivery by PEGylated drug conjugates.

The current review presents an update of drug delivery using poly(ethylene glycol) (PEG), that focuses on recent developments in both protein and organic drugs. Certainly the past 10 years has resulted in a renaissance of the field of PEG drug conjugates, initiated by the use of higher molecular weight PEGs (M(w)>20,000), especially 40,000 which is estimated to have a plasma circulating t(1/2) of approximately 10 h in mice. This recent resuscitation of small organic molecule delivery by high molecular weight PEG conjugates was founded on meaningful in vivo testing using established tumor models, and has led to a clinical candidate, PEG-camptothecin (PROTHECAN), an ester based prodrug currently in phase II trials. Additional applications of high molecular weight PEG prodrug strategies to amino containing drugs are presented: similar tripartate systems based on lower M(w) PEG and their use with proteins is expounded on. The modification of a benzyl elimination tripartate prodrug specific for mercaptans is presented, and its successful application to 6-mercaptopurine giving a water soluble formulation is discussed. Recent novel PEG oligonucleotides and immunoconjugates are also covered. Clinical results of FDA approved PEGylated proteins are also presented.

Camptothecin delivery systems : enhanced efficacy and tumor accumulation of camptothecin following its conjugation to polyethylene glycol via a glycine linker

Purpose: This study was designed to assess the circulatory retention, antitumor activity and tissue biodistribution of polyethylene glycol (PEG)-conjugated camptothecin-20-O-glycinate, PEG-β-camptothecin (PEG-β-CPT). PEG-β-CPT is a novel water-soluble transport form (macromolecular prodrug) of the naturally derived antitumor drug, 20-(S )-camptothecin (CPT). Methods: Circulatory retention studies were performed in nontumor-bearing mice injected intravenously (i.v.) with 875 mg/kg of PEG-β-CPT. Antitumor activity was evaluated both intraperitoneally (i.p.) and i.v. in nude mouse xenograft models. Biodistribution studies were performed in nude mice bearing colorectal carcinoma xenografts with tritium-labelled PEG-β-CPT and CPT injected i.v. Results: PEG-β-CPT had a blood t1/2α of approximately 6 min and a t1/2β of 10.2 h. Significant antitumor activity was seen in all treated xenograft models. Biodistribution studies demonstrated that PEG-β-CPT in saline provided more available labelled CPT in the circulation than unconjugated CPT dissolved in intralipid. In addition, it appeared that more labelled CPT accumulated in solid tumors when delivered in the PEG-β-CPT form, with greater preference for tumor tissue than normal tissue. Conclusion: This soluble transport form of CPT and its underlying technology may have clinical application especially for the treatment of solid tumors.

Camptothecin-20-PEG ester transport forms: the effect of spacer groups on antitumor activity.

An improved synthesis of the hindered PEG-camptothecin diester transport form has been achieved using the Mukaiyama reagent. We have also assessed the effect of changing the electronic configuration of the (d-position of PEG-camptothecin transport forms on the rates of hydrolysis of the pro-moiety, and attempted to correlate these differences to efficacy in two animal models. In addition to the simple substitution of N for O, other synthetic modifications of these atoms were accomplished by employing heterobifunctional linker groups. The half lives by disappearance (rates of hydrolysis) of the transport forms in buffer and rat plasma were determined. It was established that anchimeric assistance to hydrolytic breakdown of the pro-moiety occurs in a predictable manner for some of these compounds. Results for the new derivatives in a P388 murine leukemic model and HT-29 human colorectal xenograft study are also presented. The use of a glycine linker group was found to provide similar efficacy in rodent models to that of simple camptothecin 20-PEG ester, and displayed enhanced pharmacokinetics.

Hemoglobin Based Oxygen Carriers: How Much Methemoglobin is too Much?

The oxidized form of hemoglobin, methemoglobin, is unable to deliver oxygen to tissues. Hemoglobin based oxygen carriers generally lack the natural oxidative-reductive machinery present within the red blood cell that converts methemoglobin to hemoglobin. This study examines tolerable levels of methemoglobin that can be present in solutions of polyethylene glycol (PEG) conjugated bovine hemoglobin without compromising its ability to deliver oxygen. Rodents were exchange-transfused to 30% of their estimated blood volume with solutions of six grams percent PEG-hemoglobin containing varying concentrations of PEG-methemoglobin. Tissue oxygenation was measured by an oxygen dependant phosphorescence quenching method. This study also looked at the level of methemoglobin formation following a top loaded infusion of low methemoglobin containing PEG-hemoglobin. Results of the oxygenation study showed that PEG-methemoglobin levels at or below 10% did not significantly alter the ability of solutions to deliver oxygen to intestines, liver, spleen and kidney. However, PEG-methemoglobin levels greater than 10% resulted in a significant decrease in PEG-hemoglobins ability to oxygenate tissues. In addition, methemoglobin levels remain low (< 10%) for a substantial period of time following PEG-hemoglobin administration.

Anticancer drug delivery systems: multi-loaded N4-acyl poly(ethylene glycol) prodrugs of ara-C. II. Efficacy in ascites and solid tumors.

The synthesis of branched PEG (40,000) acids has been achieved using aspartic acid (Asp) and AspAsp dendrons. Complete conjugation of these dendritic acids with cytosine arabinoside (ara-C) was achieved by the use of spacers that allowed a greater separation of the branches to accommodate several large ara-C molecules in proximity to each other. The tetrameric and octameric PEG-ara-C amide prodrugs were much more effective in the treatment of solid and ascites tumors compared to the native drug. The greater loading of the PEG backbone appears to have achieved a minimum threshold concentration for the therapeutic delivery of ara-C.

Anticancer drug delivery systems: N4-acyl poly(ethyleneglycol) prodrugs of ara-C. I. Efficacy in solid tumors.

A systematic study of N(4) amino PEG-prodrugs of ara-C (1) was conducted and provided a series of disubstituted amides, as well as a carbamate derivative. These conjugates showed hydrolysis half lives in rat plasma from about 1 h to 3 days, but were stable for >24 h in phosphate buffer, pH 7.4. In an LX-1 solid lung tumor model some of the PEG prodrugs exhibited superior activity to ara-C when compared on a molar basis. One problematic issue that was identified in this investigation was the need to increase the loading of ara-C onto PEG in order to avoid highly viscous solutions.

Drug delivery of anticancer agents: water soluble 4-poly (ethylene glycol) derivatives of the lignan, podophyllotoxin.

This paper reports on the synthesis and in vivo oncolytic activity of a series of water-soluble acyl derivatives of polyethylene glycol (PEG) conjugated podophyllotoxin. Some analogs of the polymer conjugate showed significantly better activity in a murine leukemia model than native podophyllotoxin suspended in an intralipid emulsion. Additionally, when tested intravenously against a solid lung tumor (A549) model, some conjugated analogs were equivalent to the podophyllotoxin/intralipid emulsion, while those compounds demonstrating slower rates of plasma hydrolysis (in vitro) appeared to cause greater toxicity. There appeared to be an overall correlation between the in vivo antitumor activity of the conjugate and its rate of hydrolysis in vitro, with those showing faster release possessing greater antitumor activity. In conclusion, the solubilization and predictable release of podophyllotoxin from a PEG carrier was achieved and resulted in some derivatives demonstrating, at a minimum, equivalency with podophyllotoxin when administered on an equal molar basis. Further studies may be warranted to assess the PEG-conjugates pharmacokinetics and therapeutic indices in leukemic models.

The Ability of Polyethylene Glycol Conjugated Bovine Hemoglobin (PEG-Hb) To Adequately Deliver Oxygen in Both Exchange Transfusion and Top-Loaded Rat Models

The purpose of this study was to determine whether a six gram percent (g%) solution of the hemoglobin based oxygen carrier, polyethylene glycol conjugated bovine hemoglobin (PEG-Hb) could adequately deliver oxygen in both partial exchange transfusion and top-loaded rat models. This study measured tissue oxygen tension, circulatory retention and cardiovascular effects following both 30% exchange transfusion and 20 to 25 mL/kg top-loaded infusions of PEG-Hb. Oxygen delivery to rat tissues was determined using an oxygen dependent phosphorescence quenching method (Oxyspot). Telemetric intravascular blood pressure probes monitored heart rate and mean arterial pressure. In both models, six g% PEG-Hb (P50-15 torr) was shown to oxygenate tissue better than stroma-free bovine Hb (P50-26 torr), cross-linked bovine Hb (P50-48 torr) or simple plasma expanders. The mean circulatory half life of PEG-Hb was 15.0 +/- 2.3 hours and 17.4 +/- 1.6 hours for exchange transfusion and 25 mL/kg top-loaded rat models, respectively. Mean arterial pressure (MAP) in PEG-Hb treated rats was insignificantly different from sham controls undergoing a 30% exchange transfusion or following a top-loaded infusion. In conclusion, the PEG conjugated form of bovine Hb with its relatively long vascular persistence may possess characteristics that facilitate tissue oxygenation in the rat.

Transitional Vacuole Formation Following a Bolus Infusion of Peg-Hemoglobin in the Rat

This study was designed to assess the morphological effects of a bolus infusion of PEG-hemoglobin on the heart, lung, liver, spleen and kidney of laboratory rats. Of particular interest was the determination of PEG-hemoglobins potential to form vacuoles in the tissues and whether these were transitory and article specific. One hundred ten female Sprague-Dawley rats were used in this study. The first experiment determined whether vacuole formation was test article specific by infusing either stroma-free bovine hemoglobin, PEG-hemoglobin, bovine serum albumin, PEG-bovine serum albumin or free PEG. The second experiment assessed the transitory nature of vacuolization. In both experiments, unconscious rats received an intravenous top-loading (bolus) injection of test article via the tail vein. Rats were sacrificed at various time points following administration and had their tissues examined for the presence of vacuoles by light microscope morphological examination and iron staining. Formation of vacuoles appeared to be test article specific with only prolonged circulating, high solute test articles producing vacuoles. These vacuoles appeared dose responsive and transitory in nature. The vacuolization found was non-toxic and believed to be due to the known effect of lysosomal overloading following the phagocytosis of vascularly persistent high solute test articles.

EVALUATION OF THE OXYGEN DELIVERY ABILITY OF PEG- HEMOGLOBIN IN SPRAGUE-DAWLEY RATS DURING HEMODILUTION

Polyethylene glycol (PEG) conjugation allows bovine hemoglobin (Hb) to retain its oxygen delivery capability while increasing its plasma expansion capacity. To determine whether PEG-Hbs ability to sustain life is due to its oxygen delivery capability rather than its plasma expansion capacity, Sprague-Dawley rats were exchange-transfused up to an 85% hematocrit reduction with either PEG-Hb, PEG-50%-methemoglobin (PEG-mHb), PEG-carbon monoxide hemoglobin (PEG-COHb) or PEG-human serum albumin (PEG-HSA). Survival and respiratory rates were monitored during the exchange transfusion, at five minutes, 24 hours and 48 hours post operative. Rats surviving 14 days were evaluated for hematology, blood chemistry and histopathology. Rats infused with PEG-Hb had a survival rate of 100% during the transfusion and 79% at 24 hours, as compared to 24 hour survival rates of 30% for PEG-mHb, and 0% for both PEG-COHb and PEG-HSA. PEG-Hb treated rats that survived the 2 week observation period had normal hematological and blood chemistry levels and no significant morphological effects. Therefore, this study demonstrates that PEG-Hb can sustain life while similar plasma expansion agents with less oxygen delivery capability are not as effective.