Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster from the marine isolate 'Streptomyces maritimus': evidence for the derailment of an aromatic polyketide synthase.

BACKGROUND: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-beta-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain 'Streptomyces maritimus' deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides. RESULTS: A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from 'S. maritimus' has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis. CONCLUSIONS: The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.

Pharmacokinetics and tissue disposition in monkeys of an antisense oligonucleotide inhibitor of Ha-ras encapsulated in stealth liposomes.

PURPOSE: This study examined the pharmacokinetics and tissue distribution of an antisense oligonucleotide ISIS 2503, formulated in stealth (pegylated) liposomes (encapsulated) or in phosphate-buffered saline (unencapsulated). METHODS: Encapsulated or unencapsulated ISIS 2503 was administered to rhesus monkeys by intravenous infusion. The concentrations of ISIS 2503 and metabolites in blood, plasma, and tissue samples were determined by capillary gel electrophoresis. RESULTS: Plasma concentrations of encapsulated ISIS 2503 decreased mono-exponentially after infusion with a mean half-life of 57.8 hours. In contrast, the concentration of unencapsulated ISIS 2503 in plasma decreased rapidly with a mean half-life of 1.07 hours. Both encapsulated and unencapsulated ISIS 2503 distributed widely into tissues. Encapsulated ISIS 2503 distributed primarily to the reticulo-endothelial system and there were few metabolites observed. In contrast, unencapsulated ISIS 2503 distributed rapidly to tissue with highest concentration seen in kidney and liver. Nuclease-mediated metabolism was extensive for unencapsulated oligonucleotide in plasma and tissues. CONCLUSIONS: The data suggest that stealth liposomes protect ISIS 2503 from nucleases in blood and tissues, slow tissue uptake, and slow the rate of clearance from the systemic circulation. These attributes may make these formulations attractive for delivering oligonucleotides to sites with increased vasculature permeability such as tumors or sites of inflammation.

The population pharmacokinetics of amphotericin B colloidal dispersion in patients receiving bone marrow transplants.

The purpose of this study was to identify the pharmacokinetics of Amphotericin B Colloidal Dispersion in patients undergoing bone marrow transplantations with systemic fungal infections and to assess the influence of ABCD on renal function. Seventy-five patients (42 females, 33 males) with a median age of 34.5 years and median weight of 70.0 kg were enrolled in the study. The plasma concentration data was available in 51/75 patients and was best described by a two-compartment model; both plasma clearance and volume of distribution increased with escalating doses; the overall average terminal elimination half-life was 29 h. Serum creatinine values over the duration of therapy were available in 59/75 patients. Overall, there was no net change in renal function over the duration of therapy.

Reduction of the cardiotoxicity of doxorubicin in rabbits and dogs by encapsulation in long-circulating, pegylated liposomes.

The relative cardiotoxicity of pegylated (STEALTH) liposomal doxorubicin (PL-DOX; Doxil) was compared to nonliposomal doxorubicin (Adriamycin) in rabbits and dogs treated i.v. for up to 22 weeks. No histological evidence of cardiotoxicity was seen in dogs treated with placebo liposomes or PL-DOX every 3 weeks for a total of 10 doses (cumulative doxorubicin dose = 10 mg/kg) either 1 or 5 weeks post-treatment. All dogs treated with the same cumulative dose of free doxorubicin showed marked cardiotoxicity (vacuolization and myofibrillar loss in the myocardium) at both time points. In rabbits, progressive cardiomyopathy was seen in both treatment groups, but was more frequent and severe with free doxorubicin (67% of doxorubicin-treated rabbits, cumulative dose = 12 to 14 mg/kg versus 16% of PL-DOX-treated animals, cumulative dose = 14 to 21 mg/kg). Five doxorubicin-treated rabbits died of congestive heart failure or with histologic evidence of cardiotoxicity (median severity score = 6). No PL-DOX-treated rabbits died of congestive heart failure, although two animals that died early showed microscopic evidence of mild cardiotoxicity (median severity score = 2.5). Cardiotoxicity increased during the post-treatment period in both treatment groups. Rabbits received up to 50% more PL-DOX with no increase in cardiotoxicity. Thus, results in two species demonstrate that the cardiotoxicity of doxorubicin is significantly decreased when administered as PL-DOX. Significantly more PL-DOX can be given without incurring an increased risk of cardiomyopathy. Recent clinical studies have confirmed that PL-DOX is also less cardiotoxic than the same dose of unencapsulated doxorubicin in humans.

Relationship of dose intensity to the induction of palmar-plantar erythrodysesthia by pegylated liposomal doxorubicin in dogs.

The multiple dose pharmacokinetics of pegylated liposomal doxorubicin (PL-DOX), known as DOXIL (US) and CAELYX (EU), was characterized in dogs and a pharmacokinetic/pharmacodynamic model to identify a relationship between drug exposure and the probability of observing treatment-related palmar-plantar erythrodysesthesia (PPE) was developed. Twenty dogs were assigned to PL-DOX groups (2/sex/ group) that received intravenous PL-DOX doses of 0.5 mg/kg ql, 2, or 4 weeks; 1.0 mg/kg q2weeks; or 1.5 mg/kg q4weeks for 12 weeks. Blood was collected for HPLC analysis of doxorubicin concentration pre-dose and periodically up to 120 h after dosing three times during treatment. Plasma drug concentration was modeled using iterative 2-stage analysis. Dermal lesions (PPE) were scored twice weekly for six regions of each dog using a 0-6 severity scale; maximum severity was 36. PPE score data were modeled using an approach in which the % probability of PPE was related to a hypothetical effect site by a series of Hill-type functions. Pharmacokinetics were best modeled as a one-compartment open model. Vss (ml/kg), CLt (ml/hr/kg) and half-life (h) were 44.1, 1.39 and 23.1, respectively. Cmax increased linearly with dose. CLt decreased with repeated doses. 5 A two-compartment pharmacodynamic model, which correctly predicted 97% of the observed lesion severity, was developed to establish the relationship of lesion severity to dose intensity (a measure of drug exposure incorporating the effect of both dose level and dosing frequency, which can be expressed in units of mg/kg/week). The model demonstrated that maximal PPE was positively correlated with dose intensity, the major factor that affects the incidence and severity of dermal lesions. 6 The model can be used to predict acceptable dose intensities in humans utilizing body surface area conversion factors and comparative AUCs for dogs and humans. It predicts that a dose intensity of 10-12.5 mg/m2 of PL-DOX will be well tolerated in patients. The results of recent clinical studies are consistent with this prediction.

Comparative pharmacokinetics, tissue distribution, and therapeutic effectiveness of cisplatin encapsulated in long-circulating, pegylated liposomes (SPI-077) in tumor-bearing mice.

PURPOSE: The pharmacokinetics (PK), biodistribution and therapeutic efficacy of cisplatin encapsulated in long-circulating pegylated (Stealth) liposomes (SPI-077) were compared with those of nonliposomal cisplatin in two murine (C26 colon carcinoma and Lewis lung) tumor models. METHODS: In therapeutic effectiveness studies, mice bearing murine C26 or Lewis lung tumors received multiple intravenous doses of SPI-077 or cisplatin in a variety of treatment schedules and cumulative doses. In the PK and biodistribution study, mice received a single intravenous bolus injection of 3 mg/kg of either SPI-077 or cisplatin 14 days after inoculation with 10(6) C26 tumor cells. Plasma and tissues were analyzed for total platinum (Pt) content by graphite furnace (flameless) atomic absorption spectrophotometery (GF-AAS). RESULTS: Efficacy studies showed that SPI-077 had superior antitumor activity compared to the same cumulative dose of cisplatin. When lower doses of SPI-077 were compared to cisplatin at its maximally tolerated dose in Lewis lung tumors, equivalent SPI-077 antitumor activity was seen at only half the cisplatin dose. Higher cumulative doses of SPI-077 were well tolerated and had increased antitumor effect. SPI-077 PK were characterized by a one-compartment model with nonlinear (saturable) elimination, whereas cisplatin PK were described by a two-compartment model with linear elimination. SPI-077 had a 55-fold lower [corrected] volume of distribution, 3-fold higher peak plasma levels, and a 60-fold larger plasma AUC compared with cisplatin. In addition, SPI-077-treated animals displayed a 4-fold reduction in Pt delivered to the kidneys (primary target organ of toxicity) relative to cisplatin, but a 28-fold higher tumor AUC than cisplatin. CONCLUSIONS: Based on the results of our studies, encapsulation of cisplatin in long-circulating pegylated liposomes has overcome limitations experienced with other liposomal cisplatin formulations. SPI-077 has a prolonged circulation time and increased tumor Pt disposition, and its antitumor effect is significantly improved compared to cisplatin in murine colon and lung cancer models.

Comparative intravenous toxicity of cisplatin solution and cisplatin encapsulated in long-circulating, pegylated liposomes in cynomolgus monkeys.

The toxicity of cisplatin encapsulated in pegylated, long-circulating liposomes (SPI-077) was compared with nonliposomal cisplatin in male and female cynomolgus monkeys (n = 2-4 per sex per group) treated with intravenous infusions of 2.5 or 25 mg/kg SPI-077, 2.5 mg/kg cisplatin, placebo liposomes, or saline once every 3 weeks for total of five treatments. All animals survived until scheduled necropsy at 3 days after the final treatment or after a treatment-free 4-week recovery period. Emesis occurred after each treatment in all cisplatin-treated monkeys, but only once in one monkey treated with high-dose SPI-077. Dose-related mild decreases in red blood cell (RBC) count, hemoglobin, and hematocrit to or slightly below low normal range occurred in the high-dose SPI-077 and placebo liposome treatment groups after each treatment, with partial to complete recovery between treatments and no signs of correlating bone marrow toxicity. Decreases were similar in cisplatin-treated monkeys, but resolved only slightly between treatments and after the end of treatment (continuing to decrease in females) and were accompanied by bone marrow hypocellularity. Indirect, but not direct, bilirubin levels were cyclically elevated in the high-dose SPI-077 and placebo-treated animals, but not in the other treatment groups. Levels had either fully resolved or were near baseline and/or saline group values prior to the next treatment. Serum cholesterol levels were cyclically increased in SPI-077- and placebo liposome-treated animals, and minimally increased numbers of foam cells were seen in the liver, spleen, kidney, and other organs; both were considered related to the lipid dose administered. Cisplatin-treated monkeys exhibited sensory polyneuropathy and moderate irreversible toxic tubular nephrosis, but no neuropathy or nephrotoxicity was seen in either SPI-077 treatment group. Microscopically, treatment-related cell death was seen in dorsal root ganglia (DRG), affecting 15% of the cells in cisplatin-treated animals, compared to 8 and 12% in the low- and high-dose SPI-077 treatment groups. Neither drug was ototoxic. In summary, repeated administration of SPI-077 produced minimal, reversible effects related to the lipid dose administered, mostly limited to the 25 mg/kg dose group. The most notable effects in this group were cyclical decreases in hematology parameters thought to be related to increased recycling of a small fraction of RBCs and limited cell death in the DRG in the absence of any neurophysiological changes. Animals treated with a 10-fold lower dose of cisplatin (2.5 mg/kg), in contrast, exhibited myelo-, nephro-, and neurotoxicity, including sensory neuropathy, and were emetic after every dose. The SPI-077 liposomal formulation of cisplatin may provide a less toxic alternative to standard cisplatin solution.

Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes.

Immunoliposomes composed of hydrogenated soy phosphatidylcholine, cholesterol, methoxypoly(ethylene glycol)-distearoyl phosphatidylethanolamine (mPEG-DSPE), and hydrazide-PEG-DSPE (mole ratio, 57:38:3.3:1.7) linked to periodate-oxidized chimerized mouse IgG (C225, anti-human epidermal growth factor receptor) were prepared by an optimized aggregation-free procedure. The antigen-binding activity of the immunoliposomes was well preserved. When injected intravenously into naive rats, the immunoliposomes (approximately 18 IgG per 100 nm liposome) exhibited long circulation times (MRT = 8.5 h, Cl = 0.2 ml/h). Subsequent injections of the immunoliposomes into the same animals resulted in rapid clearance (MRT < or = 0.7 h, Cl > or = 7 ml/h), which was accompanied by a significant increase in anti-C225 specific titers. Upon repeated injection or coinjection with the parent liposomes free C225 consistently exhibited prolonged circulation without any increase in C225-specific antisera, but was cleared quickly when administered into animals that had been pretreated with the immunoliposomes. Screening of the immunoliposome induced antisera against human polyclonal IgG and C225-derived Fab' fragment revealed that the immune response was specifically triggered by the constant human region of C225. These results demonstrate that the preparations of PEG-grafted immunoliposomes are more immunogenic than the free IgG component, which is of profound importance to the antibody-mediated liposomal drug delivery effort.

Sterically stabilized pH-sensitive liposomes. Intracellular delivery of aqueous contents and prolonged circulation in vivo.

Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.

Insertion of poly(ethylene glycol) derivatized phospholipid into pre-formed liposomes results in prolonged in vivo circulation time.

Transfer of MPEG(1900)-DSPE from micellar phase to pre-formed liposomes imparts long in vivo circulation half-life to an otherwise rapidly cleared lipid composition. MPEG(1900)-DSPE transfers efficiently and quickly in a time and temperature dependent manner. There is negligible content leakage and a strong correlation between assayed mol% MPEG(1900)-DSPE, liposome diameter increase, and pharmacokinetic parameters such as distribution phase half-life. Since a biological attribute (liposome clearance rate) can be modified by the insertion process, it suggests a simple and economical way to impart site-specific targeting to a variety of liposome delivery systems. This method is also a convenient way to measure the 'brush' thickness of such conjugates directly.

Population pharmacokinetics and renal function-sparing effects of amphotericin B colloidal dispersion in patients receiving bone marrow transplants.

The purpose of this study was to evaluate the pharmacokinetics of amphotericin B colloidal dispersion and its effect on creatinine clearance in bone marrow transplant patients with systemic fungal infections. Seventy-five patients (42 females and 33 males) with a median age of 34.5 years and a median weight of 70.0 kg were enrolled in the study. Patients received 1 of 15 dose levels (range, 0.5 to 8.0 mg/kg of body weight) daily for a mean duration of 28 days and a mean cumulative dose amount of 8 g. Plasma samples for amphotericin B determination (median number, 4; range, 2 to 30) and daily serum creatinine values were obtained for each patient. Iterative two-stage analysis, one of several approaches to population pharmacokinetic and pharmacodynamic modelling, was employed for the pharmacokinetic analysis. The plasma data were available for 51 of 75 patients and were best described by a two-compartment model. Both plasma clearance and volume of distribution increased with escalating doses; the overall average terminal elimination half-life was 29 h. Of the covariates studied, only body weight and dose size were significant. Serum creatinine values over the duration of therapy were available for 59 of 75 patients. Overall, there was no net change in renal function over the duration of therapy; 12 patients had > 30% increases in creatinine clearance, whereas 13 had > 30% decreases. No measure of amphotericin B colloidal dispersion exposure, demographic values, or concomitant treatment with other medications was related to changes in the creatinine clearance.

Pharmacokinetics and anti-tumor activity of vincristine encapsulated in sterically stabilized liposomes.

Vincristine is used clinically for the treatment of various types of cancer. Recent significant therapeutic improvements obtained by entrapping anthracyclines in sterically stabilized liposomes raised the question whether the therapeutic index of vincristine can be similarly increased by formulation into such long-circulating liposomes. Encapsulation of vincristine in sterically stabilized liposomes (SL-VCR) prolonged the drug's distribution phase plasma half-life in rats from 0.22 to 10.5 hr. There was no significant difference in LD50 (> < or = 2.5 mg/kg, i.v.), but mice given sublethal doses of SL-VCR experienced significantly less weight loss than those given the same dose of free drug. Compared to free drug, SL-VCR was most effective against i.p. or s.c. implanted tumors. However, i.v. tumor inoculation nullified the therapeutic advantage of encapsulation. A single i.v. 2 mg/kg dose of SL-VCR increased the life span of mice bearing i.v. implanted P388 cells by only 44%, while the life span of i.p. P388 implanted mice was increased by 199%. In an s.c. implanted murine colon carcinoma, multiple doses of free drug did little to slow the growth of the tumors, but SL-VCR was able to produce long-term survivors in several dose regimens. These results indicate that prolonged circulation time increases the therapeutic index of VCR entrapped in liposomes against s.c. or i.p. implanted tumors, but does not improve the drug's activity against rapidly growing i.v. disseminated leukemias.

Long circulating, cationic liposomes containing amino-PEG-phosphatidylethanolamine.

Ligand attachment to polyethylene glycol (PEG) grafted, long circulating liposomes at the polymer terminus is of interest for targeting but the effect of positively charged groups is unknown. Amino-polyethylene glycol-phosphatidylethanolamine (AminoPEG-PE), prepared in four steps from alpha-amino-omega-hydroxy-PEG, was tested for influence on liposome interactions in vivo: blood circulation and biodistribution. Despite surface amines on each liposome conferring cationic behavior, in vivo properties are comparable to those obtained with methoxy-PEG-PE. The consequences are profound for targeting and possibly systemic delivery of cationic lipidic-polynucleotide complexes.

Transendoscopic electrocautery-induced gastric ulcers as a model for gastric healing studies in ponies.

The ponies were apparently healthy and 6-20 months of age. In Study 1, gastric lesions were created by transendoscopic electrocautery in the non-glandular gastric mucosa, adjacent to the margo plicatus in 9 ponies which were then treated with water, 12 mg cimetidine HCl/kg bwt or 18 mg cimetidine HCl/kg bwt per os every 12 h for 35 days. In Study 2, gastric lesions were similarly induced in 9 ponies in the non-glandular mucosa and also in the glandular mucosa just below the non-glandular lesion on the greater curvature of the stomach. The ponies were treated with water, 8 mg cimetidine/kg bwt or 16 mg cimetidine/kg bwt per os every 8 h for 21 days. In both studies gastric lesion healing was monitored twice weekly by video gastroscopy. There was no apparent difference in healing times between the water and cimetidine treatment groups in either study. These results indicate that uniform gastric ulcers can be created by transendoscopic electrocautery in the non-glandular mucosa of ponies and that these ulcers heal at a predictable rate which should be useful in studying compounds that might accelerate healing of gastric mucosal lesions. However, cimetidine was not effective in accelerating the rate of healing under the conditions of this study.

Sterically stabilized liposomes: physical and biological properties.

Advanced liposomal therapeutics has been attained by liposome surface modification, initially with specific glycolipids and subsequently with surface-grafted PEG, reducing in vivo rapid recognition and uptake, giving prolonged blood circulation, and providing selective localization in tumors and other pathological sites, as described in recent reviews. The result is improved efficacy of encapsulated agents. The surface PEG may produce a steric barrier, as described for colloids. Reduced in vivo uptake may result from inhibition of plasma-protein adsorption, or opsonization, by the steric coating. Several physical studies support this mechanism, including electrophoretic mobility (zeta potential). Our previous results for 2000-dalton PEG indicated a coating thickness about 5 nm, in agreement with independent measurements. We report here results for 750 to 5000-dalton PEGs. The calculated coating thickness increases with molecular weight in a nonlinear fashion. The dependence of blood circulation and tissue distribution on PEG molecular weight correlates with zeta-potential estimates of PEG-coating thickness. Effects on tissue distribution are reported for liver and spleen, the major phagocytic organs. The biological properties of these liposomes depend on the surface polymer rather than the lipid bilayer, yielding important advantages for lipid-mediated control of drug interaction and release without affecting the biodistribution.

Versatility in lipid compositions showing prolonged circulation with sterically stabilized liposomes.

Efforts to overcome rapid uptake of liposomes by cells of the mononuclear phagocytic system (MPS) have identified that lipids derivatized with the hydrophilic polymer poly(ethylene glycol) (PEG) have many advantages. The structure-function relationship of PEG-derivatized phosphatidylethanolamine (PEG-PE) has been examined by studies of blood lifetime and tissue distribution in both mice and rats. Liposomes composed of phosphatidylcholine (PC), cholesterol, and 7.5 mol% of PEG-PE show prolonged circulation and reduced MPS uptake when the PEG has a molecular weight in the range of 1000 to 5000. Up to 35% of the injected dose remains in the blood and less than 10% is taken up by the MPS (liver plus spleen) after 24 h in the best cases as compared to 1% and 40%, respectively, for liposomes without PEG-PE. Prolonged circulation with PEG-PE is independent of cholesterol, degree of saturation in either the PC or the PE lipid anchor, lipid dose, or addition of other negatively charged lipids, phosphatidylglycerol or cholesterol sulfate. This versatility in lipid composition and dose without alteration of blood lifetime or tissue distribution is essential for controlling drug dosage and release properties in a liposome-based therapeutic agent.

Prolonged systemic delivery of peptide drugs by long-circulating liposomes: illustration with vasopressin in the Brattleboro rat.

The value of novel systemically long-circulating liposomes to prolong the duration of an antidiuretic hormone, arg8-vasopressin (VP), was investigated as a representative of low molecular weight peptides with rapid clearance. Cholesterol content was found to have a controlling effect on VP release in serum. Three types of liposomes were selected for urine production measurements in VP deficient Brattleboro rats. One contained phosphatidylserine (PS), which was rapidly cleared from the circulation. In the other two liposomes, the PS component was replaced by either phosphatidylglycerol or a novel phospholipid derivatized with polyethylene glycol (PEG); both showing prolonged circulation. Free VP (up to 8 micrograms/kg) gave reduced urine production for less than 24 hr. The PG formulation exhibited a dose-dependent prolonged duration of bioactivity of up to 4 days. Substitution of PEG-PE resulted in a 2-day delay followed by a prolonged duration of bioactivity for over 4 days. The duration of the prolonged bioactivity was not dose dependent but the amplitude was. This is attributed to VP release from liposomes which have distributed intact to another compartment without having been taken up by the RES. By balancing liposome circulation time, release rate, and dose, long-circulating liposomes can be applied to prolong the biological activity of a therapeutic peptide.

Isolation and regional localization of the murine homeobox-containing gene Hox-3.3 to mouse chromosome region 15E.

A murine homeobox-containing cDNA clone has been isolated from an adult spinal cord library. Using in situ hybridization and somatic cell genetics techniques, the newly isolated homeobox gene has been mapped to mouse chromosome region 15E. Because of its chromosomal location, we called this gene locus Hox-3.3. Nucleotide sequence analysis revealed that the Hox-3.3 gene represents the murine cognate of the human homeobox gene c8. The presumptive organization of the murine Hox-3 homeobox gene cluster is discussed.

Regional localization of the interferon-beta 2/B-cell stimulatory factor 2/hepatocyte stimulating factor gene to human chromosome 7p15-p21.

The human interferon-beta 2 gene (IFNB2) is identical to the genes encoding the B-cell stimulatory factor (BSF-2), the hybridoma growth factor (HGF), and the hepatocyte stimulating factor (HSF). This protein mediates major alterations in the secretion of a wide spectrum of plasma proteins by the liver in response to tissue injury (the acute-phase response). We have used a cDNA probe specific to the human IFNB2 gene in DNA hybridization experiments and report the regional localization of this gene to human chromosome 7p15-p21. Southern blot analyses of DNA extracted from a panel of mouse X human somatic cell hybrids localized this gene to human chromosome 7p. In situ hybridization of the IFNB2 cDNA probe to prebanded human metaphase chromosome spreads allowed the further localization of this gene to 7p15-p21.