Prolonged Tissue Retention Research Articles

Supramolecular Enzyme-Peptide Gels for Localized Therapeutic Biocatalysis.

Enzyme therapeutics are often compromised by poor accumulation within target tissues, necessitating high doses that can exacerbate off-target effects. We report an injectable supramolecular enzyme-peptide gel platform for prolonged local enzyme retention in vivo . The gel is based on CATCH(+/-) ( C o- A ssembling T ags based on CH arge-complementarity), cationic and anionic peptide pairs that form β-sheet fibrils upon mixing 1 . Enzymes recombinantly fused to CATCH(-) peptide integrate into CATCH(+/-) β-sheet fibrils during assembly, resulting in an enzyme-peptide gel. Catalytically-active gels were fabricated with four disparate enzymes: CATCH(-)-NanoLuc luciferase, CATCH(-)-cutinase, CATCH(-)-uricase, and CATCH(-)-adenosine synthase A. CATCH(-)-cutinase gels demonstrated tunability of the platform, while CATCH(-)-NanoLuc gels demonstrated prolonged tissue retention relative to soluble enzyme. CATCH(-)-uricase gels resolved localized inflammation in a gout model, while CATCH(-)-adenosine synthase A gels suppressed localized lipopolysaccharide-induced inflammation. Modular and tunable enzyme content, coupled with prolonged tissue retention, establish CATCH enzyme-peptide gels as a generalizable vehicle for effective local therapeutic biocatalysis.

Nanostructured lipid carriers loaded into in situ gels for breast cancer local treatment

In this study, nanostructured lipid carriers (NLC) were developed and employed to obtain in situ thermosensitive formulations for the ductal administration and prolonged retention of drugs as a new strategy for breast cancer local treatment. NLC size was influenced by the type and concentration of the oil phase, surfactants, and drug incorporation, ranging from 221.6 to 467.5 nm. The type of liquid lipid influenced paclitaxel and 5-fluorouracil cytotoxicity, with tributyrin-containing NLC reducing IC50 values by 2.0–7.0-fold compared to tricaprylin NLC in MCF-7, T-47D and MDA-MB-231 cells. In spheroids, the NLCs reduced IC50 compared to either drug solution (3.2–6.2-fold). Although a significant reduction (1.26 points, p < 0.001) on the health index of Galleria mellonella larvae was observed 5 days after NLC administration, survival was not significantly reduced. To produce thermosensitive gels, the NLCs were incorporated in a poloxamer (11 %, w/w) dispersion, which gained viscosity (2-fold) at 37 °C. After 24 h, ∼53 % of paclitaxel and 83 % of 5-fluorouracil were released from the NLC; incorporation in the poloxamer gel further prolonged release. Intraductal administration of NLC-loaded gel increased the permanence of hydrophilic (2.2–3.0-fold) and lipophilic (2.1–2.3-fold) fluorescent markers in the mammary tissue compared to the NLC (as dispersion) and the markers solutions. In conclusion, these results contribute to improving our understanding of nanocarrier design with increased cytotoxicity and prolonged retention for the intraductal route. Tributyrin incorporation increased the cytotoxicity of paclitaxel and 5-fluorouracil in monolayer and spheroids, while NLC incorporation in thermosensitive gels prolonged tissue retention of both hydrophilic and hydrophobic compounds.

Inflammatory conditions shape phenotypic and functional characteristics of lung-resident memory T cells

Abstract Lung tissue-resident memory T cells (T RM) are critical for the local control of viral respiratory tract infections. However, the exact mechanisms of T RMstimulation and maintenance are not fully understood, rendering the induction of protective T RMresponses by vaccination an ambitious goal. Here, T RMpopulations induced by natural influenza A virus (IAV) infection or by mucosal immunization using adenoviral vector vaccines encoding the IAV antigens HA and NP as well as IL-1β as adjuvant were analyzed. Phenotypic and functional T RMresponses were assessed in mice over 200 days via flow cytometry. Interestingly, long-lasting T RMresponses with a less cytotoxic character were found scattered within the lung tissue after mucosal immunization. By contrast, T RMtriggered by prior IAV infection surrounded bronchus-associated B cell clusters and their quantity waned over time more rapidly. Furthermore, single cell RNA sequencing revealed different gene expression profiles of NP-specific T RMdepending on the priming condition. However, both T RMpopulations provided protection from heterologous challenge, resulting in an expansion of cognate antigen-specific T RM. Substantial differences in IAV-specific T-cell immunity after adenoviral vector immunization or prior infections could be confirmed in the Babraham pig model. Noticeably, increased T RMresponses did not correlate with protection against an infection with a heterologous IAV strain, indicating potentially different correlates of protection in pig and mice. In conclusion, mucosal immunization was associated with a prolonged tissue retention of functional T RM, opening up a promising line of enquiry for the design of future vaccines against respiratory viruses. Main parts of this work were supported by grants from IZKF (advanced project A90). The translational part into the pig model was carried out at the Pirbright Institute, supported by grants from VetBioNet (grant agreement No 731014, Ref: VBN_20_49) and BBSRC.

Effects of Two Soluble ACE2-Fc Variants on Blood Pressure and Albuminuria in Hypertensive Mice: Research Letter.

Angiotensin-converting enzyme 2 (ACE2) hydrolyzes angiotensin (Ang) II to Ang-(1-7), promoting vasodilatation, and inhibiting oxidative stress and inflammation. Plasma membrane ACE2 is the receptor for all known SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral variants. In COVID-19 infection, soluble ACE2 variants may act as decoys to bind and neutralize the coronavirus, reducing its tissue infectivity. Furthermore, soluble ACE2 variants have been proposed as potential therapeutics for kidney disease and hypertensive disorders. Soluble ACE2 variants conjugated to human Fc domains and selected for high-potency viral SARS-CoV-2 neutralization were prepared and evaluated for ACE2 activity in vitro. Lead candidates were then tested for systemic ACE2 activity, stability, and effects on blood pressure and albuminuria in mice with Ang II-induced hypertension. ACE2 activity of 10 soluble ACE2 variants was first assessed in cell-free conditions using a fluorogenic substrate, or by Ang II hydrolysis to Ang-(1-7). Hypertension was induced in male or female mice by implantation of osmotic minipumps containing Ang II. Two lead ACE2 variants were injected intravenously (i.v.) into hypertensive mice, followed by measurements of blood pressure (tail-cuff plethysmography), albuminuria, and tissue ACE2 activity and protein (immunoblots). Soluble ACE2-Fc variants demonstrated significant ACE2 enzymatic activity, with kinetics comparable with human recombinant ACE2. In hypertensive mice, single dose i.v. injection of ACE2-Fc variant K (10 mg/kg) significantly decreased systolic blood pressure at 24 hours, with partial lowering sustained to 48 hours, and tendency to reduce albuminuria at 72 hours. By contrast, ACE2-Fc variant I had no effect on blood pressure or albuminuria in hypertensive mice; ACE2-Fc variant K was detected by immunoblotting in plasma, kidney, heart, lung, liver, and spleen lysates 72 hours after injection, associated with significantly increased ACE2 activity in all tissues except kidney and spleen. Angiotensin-converting enzyme 2-Fc variant I had no effect on plasma ACE2 activity. Soluble ACE2-Fc variant K reduces blood pressure and tends to lower albuminuria in hypertensive mice. Furthermore, soluble ACE2-Fc variant K has prolonged tissue retention, associated with increased tissue ACE2 activity. The results support further studies directed at the therapeutic potential of soluble ACE2-Fc variant K for cardiovascular and kidney protection.

Facile preparation of Au- and BODIPY-grafted lipid nanoparticles for synergized photothermal therapy.

Gold nanoparticles with large size exhibit preferable properties for photothermal therapy (PTT). However, the prolonged tissue retention and slow elimination of gold nanoparticles limit their therapeutic applications. Previously, gold nanoclusters carrying lipid nanoparticles (Au-LNPs) have been reported after simply mixing Au3+ with preformed diethylenetriaminepentaacetic acid lipid nanoparticles to solve this contradiction. Au-LNPs demonstrated enhanced photothermal effects in comparison to neat gold nanoparticles. To further improve the photothermal activity, we introduced the organic photothermal agent boron dipyrromethene (BODIPY) to Au-LNPs for synergistic PTT. Au- and BODIPY-grafted LNPs (AB-LNPs) were formed by simply mixing Au-LNPs with BODIPY. The BODIPY could be associated stably to Au-LNPs, and the release of BODIPY from AB-LNPs could be accelerated by laser irradiation. AB-LNPs are scalable and showed excellent photothermal effects. AB-LNPs showed enhanced cellular uptake efficiency compared to free BODIPY in 4T1 breast cancer cells. Under laser irradiation, AB-LNPs exhibited synergistic photothermal effects with significantly reduced dosage compared to monotherapy (treatments with Au-LNPs or free BODIPY alone). This study thus provides a facile and adaptive strategy for the development of a scalable and safe high-performance nanoplatform for synergistic PTT in the treatment of cancer and other diseases.

If small molecules immunotherapy comes, can the prime be far behind?

Anti-cancer immunotherapy, which includes cellular immunotherapy, immune checkpoint inhibitors and cancer vaccines, has transformed the treatment strategies of several malignancies in the past decades. Immune checkpoints blockade (ICB) is the most commonly tested therapy and has the potential to induce a durable immune response in different types of cancers. However, all approved immune checkpoint inhibitors (ICIs) are monoclonal antibodies (mAbs), which are fraught with disadvantages including lack of oral bioavailability, prolonged tissue retention and poor membrane permeability. Therefore, the research focus has shifted to developing small molecule inhibitors to obviate the limitations of mAbs. Given the complexity of the tumor micro-environment (TME), the combination of ICIs with various small molecule agonists/inhibitors are currently being tested in clinical trials to improve treatment outcomes and prevent tumor recurrence. In this review, we have summarized the mechanisms and therapeutic potential of several molecular targets, along with the current status of small molecule inhibitors.

Tissue distribution of rIX‐FP after intravenous application to rodents

BackgroundHemophilia B is caused by coagulation factor IX (FIX) deficiency. Recombinant fusion protein linking coagulation FIX with recombinant albumin (rIX‐FP; Idelvion®) is used for replacement therapy with an extended half‐life. A previous quantitative whole‐body autoradiography (QWBA) study investigating the biodistribution of rIX‐FP indicated equal biodistribution, but more prolonged tissue retention compared with a marketed recombinant FIX product. ObjectivesTo complete and confirm the QWBA study data by directly measuring rIX‐FP protein and activity levels in tissues following intravenous (i.v.) administration to normal rats and FIX‐deficient (hemophilia B) mice. MethodsAfter i.v. administration of rIX‐FP at a dose of 2000 IU/kg, animals were euthanized at specific time points up to 72 hours postdosing. Subsequently, plasma and various tissues, which were selected based on the previous QWBA results, were harvested and analyzed for FIX antigen levels using an ELISA (both species) or an immunohistochemistry method (mice only), as well as for FIX activity levels (mice only) using a chromogenic assay. ResultsIn rats, rIX‐FP distributed extravascularly into all tissues analyzed (ie, liver, kidney, skin and knee) with peak antigen levels reached between 1 and 7 hours postdosing. In hemophilia B mice, rIX‐FP tissue distribution was comparable to rats. FIX antigen levels correlated well with FIX activity readouts. ConclusionsOur results confirm QWBA data showing that rIX‐FP distributes into relevant target tissues. Importantly, it was demonstrated that rIX‐FP available in tissues retains its functional activity and can thus facilitate its therapeutic activity at sites of potential injury.

Optimizing a Novel Au-Grafted Lipid Nanoparticle Through Chelation Chemistry for High Photothermal Biologic Activity

Gold nanoparticles through nucleation of Au clusters have been extensively studied. However, due to low potency, prolonged tissue retention, and irreversible accumulation, the safety considerations have limited their therapeutic and diagnostic applications. Novel gold nanostructures with retained physical properties and higher biodegradability could be prepared by alternative approaches. Previously, a lipid nanoparticle (LNP) platform carrying gadolinium (Gd3+) has been reported to eliminate through the biliary without accumulation in the liver or kidney within 24 h. Inspired by this discovery, we investigated a new approach of forming gold nanoparticles using preformed LNPs grafting diethylenetriamine-pentaacetic acid as a chelating agent. Tiny Au nanoparticles are formed by simply mixing Au3+ with preformed diethylenetriamine-pentaacetic acid–LNP. The Au3+ associates stably to these LNPs after a systematic optimization. The Au-grafted LNPs are scalable and showed excellent photothermal effects when subjected to near-infrared light irradiation. They exhibit enhanced light-induced tumor cell killing at higher efficiency, compared with that of classical gold nanoparticles (citrated reduced). Given an additional small dose (2 Gy) of gamma irradiation, Au-grafted LNP could produce synergistic photothermal and radiotherapeutic effects under reduced light dose. The simple and adaptive nanoparticle design may enhance the margin of safety of gold nanoparticles in the treatment of cancers and other diseases.

Improving Efficacy and Safety of Agonistic Anti-CD40 Antibody Through Extracellular Matrix Affinity.

CD40 is an immune costimulatory receptor expressed by antigen-presenting cells. Agonistic anti-CD40 antibodies have demonstrated considerable antitumor effects yet can also elicit serious treatment-related adverse events, such as liver toxicity, including in man. We engineered a variant that binds extracellular matrix through a super-affinity peptide derived from placenta growth factor-2 (PlGF-2123-144) to enhance anti-CD40's effects when administered locally. Peritumoral injection of PlGF-2123-144-anti-CD40 antibody showed prolonged tissue retention at the injection site and substantially decreased systemic exposure, resulting in decreased liver toxicity. In four mouse tumor models, PlGF-2123-144-anti-CD40 antibody demonstrated enhanced antitumor efficacy compared with its unmodified form and correlated with activated dendritic cells, B cells, and T cells in the tumor and in the tumor-draining lymph node. Moreover, in a genetically engineered BrafV600E βCatSTA melanoma model that does not respond to checkpoint inhibitors, PlGF-2123-144-anti-CD40 antibody treatment enhanced T-cell infiltration into the tumors and slowed tumor growth. Together, these results demonstrate the marked therapeutic advantages of engineering matrix-binding domains onto agonistic anti-CD40 antibody as a therapeutic given by tumori-regional injection for cancer immunotherapy.Implications: Extracellular matrix-binding peptide conjugation to agonistic anti-CD40 antibody enhances antitumor efficacy and reduces treatment-related adverse events. Mol Cancer Ther; 17(11); 2399-411. ©2018 AACR.

Development of Inhalable Dry Powder Formulations Loaded with Nanoparticles Maintaining Their Original Physical Properties and Functions

Functional nanoparticles, such as liposomes and polymeric micelles, are attractive drug delivery systems for solubilization, stabilization, sustained release, prolonged tissue retention, and tissue targeting of various encapsulated drugs. For their clinical application in therapy for pulmonary diseases, the development of dry powder inhalation (DPI) formulations is considered practical due to such advantages as: (1) it is noninvasive and can be directly delivered into the lungs; (2) there are few biocomponents in the lungs that interact with nanoparticles; and (3) it shows high storage stability in the solid state against aggregation or precipitation of nanoparticles in water. However, in order to produce effective nanoparticle-loaded dry powders for inhalation, it is essential to pursue an innovative and comprehensive formulation strategy in relation to composition and powderization which can achieve (1) the particle design of dry powders with physical properties suitable for pulmonary delivery through inhalation, and (2) the effective reconstitution of nanoparticles that will maintain their original physical properties and functions after dissolution of the powders. Spray-freeze drying (SFD) is a relatively new powderization technique combining atomization and lyophilization, which can easily produce highly porous dry powders from an aqueous sample solution. Previously, we advanced the optimization of components and process conditions for the production of SFD powders suitable to DPI application. This review describes our recent results in the development of novel DPI formulations effectively loaded with various nanoparticles (electrostatic nanocomplexes for gene therapy, liposomes, and self-assembled lipid nanoparticles), based on SFD.

A targeted nanoglobular contrast agent from host-guest self-assembly for MR cancer molecular imaging

The clinical application of nanoparticular Gd(III) based contrast agents for tumor molecular MRI has been hindered by safety concerns associated with prolonged tissue retention, although they can produce strong tumor enhancement. In this study, a targeted well-defined cyclodextrin-based nanoglobular contrast agent was developed through self-assembly driven by host-guest interactions for safe and effective cancer molecular MRI. Multiple β-cyclodextrins attached POSS (polyhedral oligomeric silsesquioxane) nanoglobule was used as host molecule. Adamantane–modified macrocyclic Gd(III) contrast agent, cRGD (cyclic RGDfK peptide) targeting ligand and fluorescent probe was used as guest molecules. The targeted host-guest nanoglobular contrast agent cRGD-POSS-βCD-(DOTA-Gd) specifically bond to αvβ3 integrin in malignant 4T1 breast tumor and provided greater contrast enhancement than the corresponding non-targeted agent. The agent also provided significant fluorescence signal in tumor tissue. The histological analysis of the tumor tissue confirmed its specific and effective targeting to αvβ3 integrin. The targeted imaging agent has a potential for specific cancer molecular MR and fluorescent imaging.

Recombinant fusion protein linking factor VIIa with albumin (rVIIa­FP): Tissue distribution in rats

IntroductionA novel fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) is currently undergoing clinical investigations. ObjectiveThis study was conducted to examine the biodistribution of rVIIa-FP in comparison to recombinant factor VIIa (rFVIIa). Materials and Methods[3H]-rVIIa-FP (10mgkg−1) or [3H]-rFVIIa (1.6mgkg−1) were administered intravenously to rats, followed by quantitative whole-body and knee joint autoradiography for 24 ([3H]-rFVIIa) or 240 ([3H]-rVIIa-FP) hours post-dose. Pharmacokinetic and excretion balance analyses were performed. ResultsIn contrast to [3H]-albumin, the tissue distributions of [3H]-rVIIa-FP and [3H]-rFVIIa were similar. Within the knee, both were rapidly present within synovial and mineralized regions. Importantly, rVIIa-FP- and albumin-derived radioactivity were detectable up to 72–120hours, whereas [3H]-rFVIIa signals were already close to detection limits at 24hours. The longest rVIIa-FP retention times were observed in bone marrow and endosteum, in which the retention times were up to 5 times longer for rVIIa-FP compared with rFVIIa. Up to 8hours post-dose, 100% of radioactivity was assigned to unchanged [3H]-rVIIa-FP. Elimination of both proteins occurred primarily via the urine. ConclusionsThe data suggest that the FVIIa moiety is directing rVIIa-FP’s tissue distribution while the albumin moiety is responsible for the prolonged tissue retention. Importantly, rVIIa-FP is highly concentrated and retained over a long period in the growth plate of the knee joint−a vulnerable site in haemophilia patients. Overall, these improved tissue distribution characteristics of rVIIa-FP may enhance compliance and allow a more convenient dosing frequency.

Pharmacokinetics and Applications of Magnetic Nanoparticles

Nanomaterials possess enormous potential for biomedical applications, while some of the most promising nanomaterials currently under investigation demonstrate prolonged tissue retention and contain heavy metals. This article investigates the pharmacokinetics of magnetic nanoparticles (MNPs) to help identify promising candidates with optimal pharmacokinetics and clearance from the body for biomedical use and synthesize nanomaterials ideal for biomedical use. The term 'magnetic nanoparticles' (MNPs) refers to nanoparticles with ferromagnetism. Such nanoparticles have special characteristics, and many in the field of biological field have undertaken research involving, e.g., immobilized enzymes and proteins, biological cells, macromolecular separation, drug carriers, and targeted, immune, biological sensors.

Drug-Coated Balloons for the Prevention of Vascular Restenosis

From early in the history of percutaneous cardiovascular intervention, restenosis has been one of the most important clinical and biological measures of success, and a great deal of effort has been put into understanding the mechanisms responsible.1,2 Restenosis is the result of the interaction of a variety of mechanical and biological processes that begin immediately after balloon injury, including early vessel recoil,3,4 negative vascular remodeling,5 and excessive neointimal proliferation.5–7 The most important limitations of balloon angioplasty, abrupt vessel closure resulting from elastic recoil and occlusive plaque dissection, were effectively solved by the introduction of balloon-expandable stents.8 However, in-stent restenosis (ISR), the result of the initial injury and the vascular response to the implanted metallic prosthesis leading to excessive neointimal proliferation, remained as the most important cause of failure of these devices.9,10 Drug-eluting stents (DES) effectively reduced ISR by addressing the biological mechanisms of neointimal proliferation and have become the mainstay of the interventional treatment of coronary atherosclerotic disease.11 However, the demonstrated efficacy of DES is balanced by the small but unpredictable risk of very late stent thrombosis thought to be due to delayed vascular healing resulting from either the initial antiproliferative effect (and associated late acquired incomplete stent apposition) or a hypersensitivity reaction to the drug, polymer coating, or their combination.12,13 Moreover, beyond the coronary vasculature, there has been a paucity of effective therapies to manage restenosis after intervention. In recent years, drug-coated balloons (DCBs) have emerged as a therapeutic alternative in the interventional field.14 With this technology, short-term transfer of antiproliferative drugs to the arterial wall is achieved without the requirement of an implanted drug delivery system, thus potentially reducing the untoward effects associated with polymer-based stent technologies. In small clinical randomized trials, paclitaxel-coated balloons have been shown to be …

Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation

In this study, we compared firefly luciferase (Fluc) reporter gene and superparamagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively. Rats (n = 31) underwent an intramyocardial injection of cardiomyoblasts (2 x 10(6)) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 microg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis. BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (> or =16 days) and persistent MR signal (> or =42 days). Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells.

Clearance Properties of Nano-Sized Particles and Molecules as Imaging Agents: Considerations and Caveats

Nanoparticles possess enormous potential as diagnostic imaging agents and hold promise for the development of multimodality agents with both imaging and therapeutic capabilities. Yet, some of the most promising nanoparticles demonstrate prolonged tissue retention and contain heavy metals. This presents serious concerns for toxicity. The creation of nanoparticles with optimal clearance characteristics will minimize toxicity risks by reducing the duration of exposure to these agents. Given that many nanoparticles possess easily modifiable surface and interior chemistry, if nanoparticle characteristics associated with optimal clearance from the body were well established, it would be feasible to design and create agents with more favorable clearance properties. This article presents a thorough discussion of the physiologic aspects of nanoparticle clearance, focusing on renal mechanisms, and provides an overview of current research investigating clearance of specific types of nanoparticles and nano-sized macromolecules, including dendrimers, quantum dots, liposomes and carbon, gold and silica-based nanoparticles.

Adsorptive voltametry to determine platinum levels in plasma from testicular cancer patients treated with cisplatin.

Patients cured of metastatic testicular cancer with cisplatin chemotherapy may suffer late adverse effects even after 20 years. The cause of these late adverse effects has not been elucidated yet. One cause might be prolonged tissue retention of platinum in these patients. Therefore, an extremely sensitive method for measuring platinum in plasma was used to investigate whether platinum is still detectable in plasma 10 to 20 years after cisplatin chemotherapy. High-pressure decomposition of plasma is followed by adsorptive voltametric determination of platinum, with a limit of quantification of 6 pg/g plasma. This procedure appeared suitable for the measurement of platinum in 44 former patients with platinum levels ranging from 22 to 140 pg/g plasma. This method is approximately 6000 times more sensitive than the standard flameless atomic absorption spectrophotometry (AAS) method. The platinum levels of these 44 patients were significantly elevated when compared with 20 control patients who were cured of testicular cancer but did not receive cisplatin chemotherapy (p < 0.001). There was a significant correlation between plasma platinum concentrations and follow-up time after cisplatin administration (r = -0.658, p < 0.001). This study shows that patients with testicular cancer who were treated with cisplatin can retain platinum in their body for at least 20 years. More data are needed to investigate whether there is a relation between the prolonged retention of platinum and long-term toxicity.

Preparation, purification, and characterization of a reversibly lipidized desmopressin with potentiated anti-diuretic activity.

. To prepare and characterize a reversibly lipidized dipalmitoyl desmopressin (DPP), and to compare its anti-diuretic efficacy and biodistribution with that of unmodified desmopressin (DDAVP). Dithiothreitol (DTT) was used to reduce the intramolecular disulfide bond in DDAVP, and the reduced DDAVP was treated with a thiopyridine-containing disulfide lipidization reagent, Pal-CPD. The product, DPP, was purified by acid precipitation and, subsequently, by size-exclusion chromatography. Reversed-phase HPLC was used to analyze the purity and to evaluate the hydrophobicity of the product. Mass spectrometry was employed to characterize its molecular structure. The biological activity of DPP was demonstrated by the antidiuretic effects in vasopressin-deficient Brattleboro rats. Preliminary pharmacokinetic and biodistribution studies of intravenously injected DDAVP and DPP were carried out in CF-1 mice. DDAVP was readily reduced by a 2-fold molar excess of DTT at 37 degrees C for 0.5 hr. DPP was formed by the reaction of reduced DDAVP with Pal-CPD. Each DPP molecule contains two palmitic acid moieties, which link to the peptide via two disulfide bonds. After acid precipitation and size-exclusion chromatography, the purity was found to be approximately 95%, and the overall yield was 57%. When DPP was administered subcutaneously to Brattleboro rats, the potency of the anti-diuretic activity of DDAVP was enhanced to more than 250-fold. The plasma concentration of intravenously injected DDAVP in mice decreased rapidly during the first 20 min and followed by a slow elimination rate. However, in DPP administered mice, the plasma concentration actually increased in the first 20 min, followed by a slow elimination with a rate similar to that in DDAVP-injected mice. The regeneration of DDAVP was detected in the plasma of mice treated with DPP. Studies of the organ distribution in mice indicated that the liver retention of DPP was longer than that of DDAVP. On the other hand, the intestinal excretion of DPP was significantly less than that of DDAVP. The 250-fold increase of the anti-diuretic potency in DPP is most likely due to a slow elimination and prolonged tissue retention, together with the regeneration of active DDAVP, in the animals. Our results indicate that reversible lipidization is a simple and effective approach for improving the efficacy of many peptide drugs.

Actinomycin and antitumor antibiotics.

Actinomycin D forms a stable complex with DNA and thereby inhibits theDNA-dependentsynthesisof RNA. The biochemical effectsof this antibiotic upon cells in culture are interpretable in terms of the complex described above. They include inhibition of the synthesis of ribosomal RNA at low drug concentrations, of all RNA species at moderate concentrations and of both RNA and DNA at very high concentrations. Given in adequate quantity, actinomycin D will inhibit synthesis of RNA in intact animals; the effect seen in a given tissue will depend upon the drug concentration achieved therein. Cytotoxicity is seen primarily in proliferating cells; it correlates better with prolonged tissue retention of the drug than with an acute inhibitory effect upon RNA metabolism. The factors responsible for selective retention of the drug within a tissue remain to be determined. Other antibiotics which may be grouped by chemical structure as anthracyclines and chromomycins also form stable complexes with DNA and inhibit synthesis of RNA.