A recombinant human enzyme for enhanced interstitial transport of therapeutics.

Subcutaneously injected therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets. This complex, three-dimensional structure limits the type and quantity of drugs that can be administered by local injection. Here we found that depolymerization of the viscoelastic component of the interstitial matrix in animal models with a highly purified recombinant human hyaluronidase enzyme (rHuPH20) increased the dispersion of locally injected drugs, across a broad range of molecular weights without tissue distortion. rHuPH20 increased infusion rates and the pattern and extent of appearance of locally injected drugs in systemic blood. In particular, rHuPH20 changed the pharmacokinetic profiles and significantly augmented the absolute bioavailability of locally injected large protein therapeutics. Importantly, within 24 h of injection, the interstitial viscoelastic barriers were restored without histologic alterations or signs of inflammation. rHuPH20 may function as an interstitial delivery enhancing agent capable of increasing the dispersion and bioavailability of coinjected drugs that may enable subcutaneous administration of therapeutics and replace intravenous delivery.

Pulmonary delivery of drugs for bone disorders.

As the population ages, osteoporosis becomes a growing public health concern. Current treatments provide patients with limited clinical improvement, numerous side effects, and no cure. The naturally-occurring peptides calcitonin and parathyroid hormone, which regulate bone metabolism, offer alternative treatment options. Clinical studies indicate the usefulness of calcitonin and parathyroid hormone in osteoporosis and Paget's disease of bone. For the peptides to become viable therapies, formulations must be developed that bypass the need for injection. Pulmonary delivery of calcitonin and parathyroid hormone appears likely in the near future.

Polycomb group repression is blocked by the Drosophila suppressor of Hairy-wing [su(Hw)] insulator.

The suppressor of Hairy-wing [SU(HW)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the SU(HW) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. In these studies, we address whether repression resulting from the Polycomb group genes can be blocked by the SU(HW) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group Response Element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. We demonstrate that the SU(HW) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome.

Developmental analysis of the ovarian tumor gene during Drosophila oogenesis.

Severe alleles of the ovarian tumor (otu) and ovo genes result in female sterility in Drosophila melanogaster, producing adult ovaries that completely lack egg chambers. We examined the developmental stage in which the agametic phenotype first becomes apparent. Germ cell development in embryos was studied using a strategy that allowed simultaneous labeling of pole cells with the determination of embryonic genotype. We found that ovo- or otu- XX embryonic germ cells were indistinguishable in number and morphology from those present in wild-type siblings. The effects of the mutations were not consistently manifested in the female germline until pupariation, and there was no evidence that either gene was required for germ cell viability at earlier stages of development. The requirement for otu function in the pupal and adult ovary is supported by temperature-shift experiments using a heat-inducible otu gene construct. We demonstrate that otu activity limited to prepupal stages was not sufficient to support oogenesis, while induction during the pupal and adult periods caused suppression of the otu mutant phenotype.

The somatic sex determines the requirement for ovarian tumor gene activity in the proliferation of the Drosophila germline.

Gametogenesis in Drosophila requires sex-specific interactions between the soma and germline to control germ cell viability, proliferation, and differentiation. To determine what genetic components are involved in this interaction, we examined whether changes in the sexual identity of the soma affected the function of the ovarian tumor (otu) and ovo genes. These genes are required cell autonomously in the female germline for germ cell proliferation and differentiation. Mutations in otu and ovo cause a range of ovarian defects, including agametic ovaries and tumorous egg cysts, but do not affect spermatogenesis. We demonstrate that XY germ cells do not require otu when developing in testes, but become dependent on otu function for proliferation when placed in an ovary. This soma-induced requirement can be satisfied by the induced expression of the 98 x 10(3) M(r) OTU product, one of two isoforms produced by differential RNA splicing. These results indicate that the female somatic gonad can induce XY germ cells to become 'female-like' because they require an oogenesis-specific gene. In contrast, the requirement for ovo is dependent on a cell autonomous signal derived from the X:A ratio. We propose that differential regulation of the otu and ovo genes provides a mechanism for the female germline to incorporate both somatic and cell autonomous inputs required for oogenesis.

Drug delivery via the respiratory tract.

Inhalation offers an enormous absorptive surface area for rapid drug absorption and substantial absorption of polypeptides. Due to slow clearance from the lower lung, even compounds with very small absorption rates can be absorbed in significant quantities over 10-12h periods. Aerosol dosimetry problems can also be minimized when lung-normal patients are considered. In the near future, optimal formulations will be combined with modified aerosol delivery devices to achieve reproducible dosing. These will be used as alternatives to parenteral delivery for drug doses of the order of milligrams or less. Research on the molecular structural dependence of lung disposition is in its infancy. Absorption kinetics for small molecules are known to depend on lipophilicity and molecular size. For macromolecules however, electronic charge and site of deposition may be additional determinants of bioavailability. Carrier-mediated absorption processes may also be important. The pulmonary absorption of a number of molecules is reviewed with special emphasis on new and promising products of biotechnology like human insulin and human growth hormone. Delivery improvements in the future should ensure, ideally, that nondenatured, monomeric pure compounds are delivered reproducibly and predominantly to the lung itself, so that these compounds may elicit reproducible systemic effects following absorption.

Genetic and molecular characterization of P element-induced mutations reveals that the Drosophila ovarian tumor gene has maternal activity and a variable null phenotype.

The mutations in the ovarian tumor (otu) gene arrest oogenesis at several stages in development. A series of deletion mutations in the otu region were characterized, each of which causes the absence or reduction of the otu transcript. These alleles range from the most severe class, which results in ovaries lacking egg cysts, to relatively mild mutations that allow the development of late stage oocytes. Heteroallelic combinations of these mutations demonstrate that the phenotypic complexity of otu mutant ovaries is due to a dosage dependent requirement for otu activity. Reciprocal cross and developmental Northern blot studies suggest a maternal requirement for otu in the development of the female germline. In addition we demonstrate that the otu zygotic null phenotype is variable, ranging from the absence of cysts in the most extreme cases, to the presence of tumorous egg chambers.

The pattern of lung injury induced after pulmonary exposure to tumor necrosis factor-alpha depends on the route of administration.

TNF-alpha is a protein elaborated by monocytes and macrophages in response to endotoxin. The in vivo consequences of TNF-alpha elaboration have been examined extensively after intravenous administration of TNF-alpha. Substantially less is known about the effects of TNF-alpha that may be generated locally by resident tissue phagocytes. We investigated the direct effects of TNF-alpha on lung tissue by administering large amounts of human TNF-alpha directly to the lung, either as an aerosol or as an intratracheal bolus. Rats were exposed to an aerosol containing several concentrations of TNF-alpha, resulting in retention of significant quantities of TNF-alpha. The histologic response to inhaled TNF-alpha was characterized by adherence of leukocytes to venular endothelium, endothelial cell disruption, and bronchovascular edema. After aerosol administration, however, there was no evidence of alveolar inflammation or edema. In contrast, intravenous administration of large amounts of human TNF-alpha, at a dose that produced a lung content of TNF-alpha similar to that produced after high-concentration aerosol exposure, resulted in severe alveolar injury and edema. Intravenous administration of TNF-alpha did not result in the bronchovascular changes seen after inhalation. To ensure that sufficient quantities of TNF-alpha were being delivered to the lung, TNF-alpha was given as an intratracheal bolus to rats. This led to measurable absorption, but the spectrum and severity of lung injury was similar to the group that received TNF-alpha as an aerosol. We conclude that in rats, the pulmonary response to the injurious effects of TNF-alpha differ, depending on whether the TNF-alpha is delivered to the air or blood side of the alveolar capillary barrier.

Inhibition of lipolysis by hydrocarbons and fatty alcohols.

The hydrolysis of long-chain triglyceride by pancreatic lipase (EC 3.1.1.3) is inhibited by hydrophobic solutes that are dissolved in the fat. Solutes tested included n-alkanes (C10-C16), aromatic and chlorinated aromatic hydrocarbons (including a PCB and DDT), n-alcohols (C10-C16), and cholesterol. Except for cholesterol, which stimulated lipolysis at low concentrations, all compounds produced roughly similar inhibition curves that followed the pattern of a typical Langmuir adsorption isotherm (Mattson, F. H., R. A. Volpenhein, and L. Benjamin, 1970. J. Biol. Chem. 245: 5335-5340). According to this interpretation, hydrophobic solutes dissolved within fat droplets partition between the interior oil phase and the surface monolayer where lipolysis occurs. Although the aromatic and chlorinated aromatic hydrocarbons were approximately 25% more inhibitory than the long-chain aliphatic hydrocarbons, as a single class, hydrocarbons were 7-10 times weaker inhibitors of lipolysis than fatty alcohols. In contrast to the alcohols whose inhibitory action may involve several mechanisms, the hydrocarbons behaved like simple dilution inhibitors; i.e., at 50% inhibition the mass ratio of hexadecane to triglyceride was 0.42. The lack of a chain length effect indicates that the hydrocarbons are not adsorbed at the interface but interdigitate the triglyceride molecules and align parallel to the lipid acyl chains. Inhibition by hydrophobic solutes was not reversed by the presence of 4 mM taurodeoxycholate and pancreatic procolipase or colipase.

Recombinant tumor necrosis factor-alpha chronically administered in rats: lack of cachectic effect.

Recombinant human tumor necrosis factor-alpha (rHuTNF) was injected into rats to test its reported cachectic effects. Rats were subcutaneously injected daily at 1730 hr with either saline or rHuTNF (0.25 mg/kg body wt) for either 5 or 14 days. Daily food intakes were significantly depressed only for the first day and first two days of rHuTNF injection in animals treated for 5 days and 14 days, respectively. There were no significant differences in daily body weights among the groups. Analysis of carcass composition revealed no significant differences in percentage of lipid or protein. Liver and inguinal pad weights were not significantly different. In vitro determination of lipogenesis showed it was enhanced in the inguinal pad and depressed in the liver only after 14 days of treatment. These results demonstrate that although in vivo rHuTNF may specifically alter tissue metabolism, it does not, by itself, result in a sustained cachectic effect.

Recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs.

To determine whether recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) prevents cough induced by exogenously applied and endogenously released neuropeptides, we measured cough responses to aerosolized solutions of substance P or of capsaicin for 2 min in random-source guinea pigs before or after exposing them to aerosolized recombinant human enkephalinase. Substance P (10(-16) M) increased coughing compared with its vehicle. Enkephalinase (120 micrograms) inhibited cough induced by subsequent exposure to substance P compared with the response to substance P alone, but after further exposure to the enkephalinase inhibitor leucine-thiorphan (10(-5) M), substance P increased cough significantly. Similar results were obtained for capsaicin-induced cough. In pathogen-free guinea pigs, after they inhaled inactive recombinant enkephalinase (33 micrograms), capsaicin (10(-13) M) increased cough significantly. In contrast, after they inhaled active recombinant enkephalinase (33 micrograms), capsaicin increased cough only slightly. These results suggest that aerosolized enkephalinase reaches the sites of release or actions of endogenous neuropeptides and, by degrading them, prevents cough induced by their release. Furthermore, these studies suggest that recombinant enkephalinase might be useful in the treatment of cough and other symptoms of diseases involving peptides cleaved by this enzyme.

Early metabolic response to tumor necrosis factor in mouse sarcoma: a phosphorus-31 nuclear magnetic resonance study.

To investigate the effects of recombinant human tumor necrosis factor alpha (rHuTNF-alpha) on high-energy phosphate metabolism of cancer cells, 31P nuclear magnetic resonance (NMR) studies were performed on a murine methylcholanthrene-induced sarcoma. Injection of 15 micrograms of rHuTNF-alpha caused progressive depletion of ATP and phosphocreatine within 90 min, together with an increase in inorganic phosphate. Metabolic changes were correlated with the early histological appearance of thrombosis and hemorrhage. A spatially localized NMR technique demonstrated that these changes were specific for the tumor. Acute ischemia of the tumor produced similar metabolic changes; thus the metabolic effects of rHuTNF-alpha could be due to either a primary action on tumor biochemistry or a secondary action produced by ischemia. These findings indicate that rHuTNF-alpha has a very rapid onset of action, which can be detected by 31P NMR. Furthermore, the results suggest that 31P NMR spectroscopy will be extremely useful for detecting early biochemical changes produced by rHuTNF-alpha or other treatments in animal and human cancers.

Human immunodeficiency virus-infected T cells and monocytes are killed by monoclonal human anti-gp41 antibodies coupled to ricin A chain.

Two human monoclonal antibodies specific for the envelope glycoprotein (gp), gp41, of the human immunodeficiency virus were conjugated to deglycosylated ricin A chain. These immunotoxins killed human immunodeficiency virus-infected H9 (T cell) and U937 (monocyte) cell lines but were nontoxic to the uninfected cell lines or to class II-positive Daudi cells. Specific killing of infected H9 cells could be completely blocked by recombinant gp160 and partially blocked by unconjugated anti-gp41 antibody but was not blocked by recombinant gp120 or human IgG demonstrating specificity for gp41. The specific toxicity of the immunotoxins for infected U937 cells was markedly potentiated by chloroquine.

Absorption of human growth hormone from the rat lung.

Recombinant methionyl human growth hormone (hGH) was administered intratracheally to adult rats, and serum concentrations of immunoreactive hGH were measured for up to 24 h. The mean absolute bioavailability was approximately 36% after 18h and was similar for doses of 0.75, 1.5, and 3 mg/kg. Peak serum hGH concentrations occurred at approximately 6 h after dosing. Tritiated hGH (3H-hGH) was used to follow the clearance of hormone from the lungs. Disappearance was linear with time and by 24 h approximately 70% of the radioactivity was gone from the lungs (elimination half-life = approximately 10.5h). Monomeric and aggregated hGH appeared to account for the majority of the residual 30% of radioactivity. Immunohistochemical localization of hGH in the alveoli suggested that the hormone was concentrated in a thin layer at the air-epithelial boundary. Pulmonary macrophages, which also stained for hGH, probably degrade hGH and thus account for some loss of material in the lungs. These studies suggest that the lung may be an alternative route for systemic delivery of recombinant proteins which are currently delivered by injection.

Growth hormone treatment reduces total body fat accumulation in Zucker obese rats.

Lean and obese Zucker rats were injected daily intraperitoneally with high doses (5-10 mg/kg) of human growth hormone (GH) for 3 weeks. In the obese rats after GH treatment, carcass lipid was decreased by 50 percent, and bone weight increased to levels of lean controls. During the last two weeks of GH treatment, food intake was increased in lean rats and not significantly affected in obese rats. Loss of body weight in obese animals was masked by water retention. Serum insulin concentrations were doubled in obese animals but unchanged in lean phenotypes after GH treatment. Hepatic fatty acid oxidation in obese animals was stimulated 5-fold by treatment, while hepatic lipid synthesis was stimulated 2-fold and adipose lipid synthesis was reduced 3-fold. These results suggest that growth hormone induces a partitioning of nutrients in obese rats which results in less lipid accumulation.

Induction of monooxygenase activity in the intestine of spot (Leiostomus xanthurus), a marine teleost, by dietary polycyclic aromatic hydrocarbons.

The response of intestinal monooxygenases to dietary polycyclic aromatic hydrocarbon (PAH) exposure was evaluated in spot (Leiostomus xanthurus), a marine teleost fish. Ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) activities were highest in the pyloric caeca and in the proximal half of the intestine. Intestinal microsomes from fish given control diets had very low levels of EROD and AHH activities relative to those in liver. After exposure to a diet containing 10 mg of 3-methylcholanthrene/kg of food, the levels of intestinal EROD and AHH activities increased 36-fold and 17-fold, respectively, such that intestinal monooxygenase activity exceeded that of the liver, which was not induced by this treatment. A significant increase in intestinal monooxygenase activity occurred in fish receiving dietary benzo[a]pyrene (BP) at concentrations as low as 10 micrograms of BP/kg food. A 5-fold increase in intestinal AHH and EROD activities was observed within 3 hr after administration of dietary BP. A plateau in gut monooxygenase activity occurred after approximately 3 days of PAH exposure; these activities decreased to control levels within 3 days after replacing the PAH diet with the control diet. Starvation resulted in disappearance of detectable monooxygenase activity. Monoclonal antibody (MAB 1-12-3) against the major PAH-inducible cytochrome P-450 (P-450E) in the liver of the marine teleost (Stenotomus chrysops) [Park et al. Arch. Biochem. Biophys. 249, 399 (1986)] recognized a single protein band in intestinal microsomes, with Mr near 54,000, which we conclude is the spot counterpart to cytochrome P-450E.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung-specific delivery of cytokines induces sustained pulmonary and systemic immunomodulation in rats.

The recombinant cytokines IFN-gamma and TNF-alpha stimulate several macrophage-mediated functions important in host defense. However, systemic administration of cytokines may be limited by significant host toxicity. We investigated whether aerosolized cytokines can stimulate alveolar macrophage and blood monocyte function, and whether they induce an inflammatory response in the lungs of normal rats. We found that aerosolized murine rIFN-gamma or recombinant human TNF-alpha increased IL-1 production by both alveolar macrophages and blood monocytes for at least 5 days after administration. Furthermore, murine rIFN-gamma increased the expression of Ia Ag on alveolar macrophages and human rTNF-alpha increased alveolar macrophage- and blood monocyte-mediated tumor lysis. Sequential aerosolization of IFN-gamma and TNF-alpha significantly increased both IL-1 release and Ia expression compared to either cytokine administered alone. Aerosolized human rTNF-alpha achieved lung levels comparable to those produced by an i.v. TNF-alpha dose reported to cause diffuse organ injury and death in rats. However, plasma TNF-alpha levels were several thousand-fold lower after aerosol administration. Aerosolized cytokines did not induce lung edema or an inflammatory cell infiltrate within the airways or alveoli. Aerosolized human rTNF-alpha alone, or murine rIFN-gamma and human rTNF-alpha, induced margination of leukocytes in pulmonary blood vessels 1 day after aerosolization, and a few small foci of pulmonary hemorrhage 5 days later. We conclude that aerosol administration of IFN-gamma or TNF-alpha enhances both pulmonary and systemic monocyte function, and that the combination of IFN-gamma and TNF-alpha produce additive or synergistic effects. Aerosolized cytokines induce only a minimal pulmonary inflammatory response. Aerosolized TNF-alpha produces high cytokine levels in the lung but very low uptake into the circulation.