Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.

Keratinocyte growth factor (KGF) stimulates the proliferation and differentiation of epithelial cells including those of the gastrointestinal tract. Although chemotherapeutics and radiation exposure kill rapidly proliferating tumor cells, rapidly dividing normal cells of the host's gastrointestinal tract are also frequently damaged, leading to the clinical condition broadly termed "mucositis." In this report, recombinant human KGF used as a pretreatment in several mouse models of chemotherapy and/or radiation-induced gastrointestinal injury significantly improved mouse survival. Using multiple-dose 5-fluorouracil, methotrexate, and radiation in combination and total body radiation alone models, KGF increased survival by 55% or greater. In the models that used chemotherapy with or without radiation, KGF significantly ameliorated weight loss after injury and accelerated weight gain during recovery. The basis of these systemic benefits appears to be due in part to the trophic effects of the growth factor on the intestinal epithelium because KGF pretreatment caused an increase in measures of mucosal thickness (villus height and crypt depth) that persisted during the course of 5-fluorouracil chemotherapy. Treatment with KGF also afforded a 3.5-fold improvement in crypt survival in the small intestine, suggesting that KGF also has a direct effect on the crypt stem cells. These data indicate that KGF may be therapeutically useful to lessen the intestinal side effects of current cancer therapy regimens.

Enteral bioavailability of human granulocyte colony stimulating factor conjugated with poly(ethylene glycol).

PURPOSE: The focus of this paper is to demonstrate that pegylation of a therapeutic protein, recombinant human granulocyte colony stimulating factor (PEG-G-CSF), results in an increase in stability and in retention of in vivo bioactivity when administered by the intraduodenal route and may, therefore, be a suitable form of the protein for inclusion in an oral delivery formulation. METHODS: The ability of PEG-G-CSF to elicit a therapeutic response from the enteral route was investigated by two methods of intraduodenal dosing in an in vivo model to determine the optimal dosing method: by slow, constant infusion, or by a single bolus administration. RESULTS: Circulating levels of the proteins confirmed that PEG-G-CSF was delivered into the systemic circulation from the enteral route and that biological activity was retained. Bioavailability from the enteral route by the constant infusion method was calculated from the intravenous administration of the proteins to be between 1.8 and 3.5% while un-modified G-CSF failed to elicit a quantifiable response by this method. Bolus administration of PEG-G-CSF also resulted in biological activity although responses were short lived and significantly lower than with the pegylated formulation. CONCLUSIONS: The possible mechanisms of enteral delivery of PEG-G-CSF are discussed. Our results indicate that oral delivery of pegylated G-CSF may be possible and in fact, preferable to using the un-modified form of the therapeutic.

Prolonged circulation of recombinant human granulocyte-colony stimulating factor by covalent linkage to albumin through a heterobifunctional polyethylene glycol.

PURPOSE: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was covalently conjugated to both rat and human serum albumin (RSA and HSA respectively) to increases the circulating half life (t1/2) of rhG-CSF. METHODS: Conjugates of RSA (MW 67,000) and HSA (MW 66,000) were prepared by linking the two proteins through a heterobifunctional maleimido-carboxyl polyethylene glycol (PEG) and were tested in the rat. The conjugates were injected intravenously (IV) at the equivalent dose of 50 micrograms/kg of rhG-CSF, and white blood cell (WBC) counts and plasma concentrations of drug were determined. A comparison of pharmacokinetic parameters was made between rhG-CSF, the conjugates RSA-PEG-rhG-CSF and HSA-PEG-rhG-CSF, and a non-covalent mixture of rhG-CSF and HSA. RESULTS: The albumin-rhG-CSF conjugates are eliminated more slowly from the circulation. The clearance values are reduced from 0.839 +/- 0.121 ml/min/kg for rhG-CSF to 0.172 +/- 0.013 ml/min/kg for RSA-PEG-rhG-CSF and 0.141 +/- 0.005 ml/min/kg for HSA-PEG-rhG-CSF. WBC counts increased in both absolute number and duration as compared to rhG-CSF alone. The albumin rhG-CSF conjugates had enhanced serum stability relative to free rhG-CSF. The rate of degradation of the albumin conjugates incubated in rat serum at 37 degrees C decreased five fold. CONCLUSIONS: The results from the study show that specific conjugation of rhG-CSF to albumin decreases plasma clearance in vivo, causes increased WBC response, and increases serum stability as compared to free rhG-CSF.

Systemic absorption and activity of recombinant consensus interferons after intratracheal instillation and aerosol administration.

PURPOSE: The pulmonary pharmacokinetics and bioactivity of E. coli derived recombinant consensus interferon (CIFN) and a modified lactose-conjugated consensus interferon (LacCIFN) were evaluated in animals. METHODS: Estimated doses of 20 and 100 micrograms/kg of the interferons were administered to anesthetized rats by aerosol via ultrasonic nebulizer as well as intravenous injection. Rats also received nominal doses of 50 and 200 micrograms/kg via intratracheal instillation (IT). Hamsters were treated with interferon via various routes including IT. The effectiveness of treatment was assessed by the resistance to development of hind leg paralysis following infection with encephalomyocarditis virus. RESULTS: Significant amounts of CIFN and LacCIFN were found in rat plasma after aerosol administration. Peak plasma levels occurred approximately 25-30 minutes with estimated bioavailabilities approaching 70%. Absorption of CIFN was rate limiting and plasma levels were detectable 12 hr post-dose. The CIFN at IT doses as low as 5 micrograms/kg was effective at reducing paralysis in hamsters but protection was variable and doses of up to 100 micrograms/kg were not 100% effective. CONCLUSIONS: Despite the incomplete protection, the results demonstrate the feasibility of treating systemic viral infections with interferon administered directly to the lungs.

A method for preparing chelate-cytokine conjugates with retention of protein structure, biological activity, and pharmacokinetic properties.

The chelating agent diethylenetriaminepentaacetic acid (DTPA) has been conjugated site-specifically to the N-terminus of recombinant human interleukin-2 (rhIL-2) by reaction with DTPA dianhydride at an initial pH of 6.0, thus demonstrating broader application of the conjugation method previously described for the structurally related cytokine rhG-CSF (Ralph et al., 1995). Purity of the DTPA-rhIL-2 conjugate, isolated by cation-exchange FPLC, and chelation of 111In were revealed by cation-exchange HPLC. Purity of the conjugate as well as chelation of radiometal were also demonstrated by SDS-PAGE and TLC, respectively. The stoichiometric molar ratio of DTPA to protein for the conjugate was approximately 1:1 as determined by TLC and mass spectrometry. Localization of the DTPA moiety was resolved by a peptide mapping procedure. The protein retained > 95% secondary structure (alpha helicity) following the conjugation. Addition of metal induced an approximate 22% loss of secondary structure for the conjugate. The in vitro biological activity of the protein was unaffected by the conjugated DTPA, even with chelated metal. Pharmacokinetic analysis of DTPA-conjugated cytokines, following chelated 111In, showed clearance and pharmacokinetic parameter values comparable to those of the corresponding unmodified cytokine. DTPA-conjugated cytokines may prove useful in cytokine research, and furthermore may represent a novel class of molecules for imaging, diagnosing, and/or treatment of malignancies where the cytokine receptor is overexpressed.

The pulmonary absorption of aerosolized and intratracheally instilled rhG-CSF and monoPEGylated rhG-CSF.

PURPOSE: The objective of this study was to highlight differences in the pulmonary absorption of a monoPEGylated rhG-CSF and rhG-CSF after intratracheal instillation and aerosol delivery. METHODS: Male Sprague Dawley rats (250 g) were anesthetized and intratracheally instilled (IT) with protein solution or were endotracheally intubated and administered aerosol for 20 min via a Harvard small animal ventilator. A DeVilbiss "Aerosonic" nebulizer containing 5 ml of protein solution at approximately 3 mg/ml was used to generate aerosol. The volume of protein solution deposited in the lung lobes was estimated to be approximately 13 microliters after delivery of Tc-99m HSA solutions. The PEGylated proteins consisted of a 6 kDa (P6) or 12 kDa PEG (P12) linked to the N-terminus of rhG-CSF. rhG-CSF also was administered IT in buffers at pH 4 and pH 7 and in dosing volumes ranging from 100 to 400 microliters. Blood samples were removed at intervals after dosing and the total white blood cell counts (WBC) were determined. Plasma was assayed for proteins by an enzyme immuno assay. RESULTS: The plasma protein concentration v. time profiles were strikingly different for aerosol v. IT delivery. The Cmax values for rhG-CSF and P12 after aerosol delivery were greater than found after IT (Aerosol: 598 +/- 135 (ng/ml) rhG-CSF; 182 +/- 14 P12 v. IT: 105 +/- 12 rhG-CSF; 65.9 +/- 5 P12). Similarly, Tmax was reached much earlier after aerosol administration (Aerosol: 21.7 +/- 4.8 (min) rhG-CSF; 168 +/- 31 P12 v. IT: 100 +/- 17 rhG-CSF; 310 +/- 121 P12). Estimated bioavailabilities (F(lung)%) were significantly greater via aerosol delivery than those obtained after IT (Aerosol: 66 +/- 14 rhG-CSF; 12.3 +/- 1.9 P12 v. IT: 11.9 +/- 1.5 rhG-CSF; 1.6 +/- 0.1 P12). An increase in circulating WBC counts was induced by all proteins delivered to the lungs. The rate and extent of absorption of rhG-CSF was not influenced by the pH employed nor the instilled volume. CONCLUSIONS: Estimates of bioavailability are dependent upon the technique employed to administer drug to the lungs. Aerosol administration provides a better estimate of the systemic absorption of macromolecules.

Lipoprotein-mediated distribution of N-aspartyl chlorin-E6 in the mouse.

The localization of many photosensitizing agents has been attributed to distribution of low density lipoprotein (LDL)-bound drug as a function of the relative numbers of LDL receptors in different tissues. While the chlorin derivative NPe6 is a potent photosensitizing agent in the mouse, it binds mainly to mouse plasma high density lipoproteins (HDL) and albumin, with only 1% bound to LDL. This pattern suggests only a minor role for the LDL-receptor pathway with regard to N-aspartyl chlorin e6 (NPe6) biodistribution. Moreover, patterns of accumulation of radioactive NPe6, LDL and HDL in murine tissues are consistent with the suggestion that distribution of NPe6 to different tissues cannot be explained on the basis of an LDL-mediated mechanism.