Long-Acting Poly(ADP-ribose) Polymerase Inhibitor Prodrug for Humans.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have been approved for once or twice daily oral use in the treatment of cancers with BRCA defects. However, for some patients, oral administration of PARPi may be impractical or intolerable, and a long-acting injectable formulation is desirable. We recently developed a long-acting PEGylated PARPi prodrug, PEG∼talazoparib (TLZ), which suppressed the growth of PARPi-sensitive tumors in mice for very long periods. However, the release rate of TLZ from the conjugate was too fast to be optimal in humans. We prepared several new PEG∼TLZ prodrugs having longer half-lives of drug release and accurately measured their pharmacokinetics in the rat. Using the rates of release of TLZ from these prodrugs and the known pharmacokinetics of free TLZ in humans, we simulated the pharmacokinetics of the macromolecular prodrugs and released TLZ in humans. From several possibilities, we chose two conjugates that could be administered intravenously every 2 weeks and maintain TLZ within its known therapeutic window. We describe situations where the PEG∼TLZ conjugates would find utility in humans and suggest how the intravenously administered long-acting prodrugs could in fact be more effective than daily oral administration of free TLZ.

A Very Long-acting Exatecan and Its Synergism with DNA Damage Response Inhibitors.

Exatecan (Exa) is a very potent inhibitor of topoisomerase I and anticancer agent. It has been intensively studied as a single agent, a large macromolecular conjugate and as the payload component of antigen-dependent antibody-drug conjugates. The current work describes an antigen-independent conjugate of Exa with polyethylene glycol (PEG) that slowly releases free Exa. Exa was conjugated to a 4-arm 40 kDa PEG through a ß-eliminative cleavable linker. Pharmacokinetic studies in mice showed that the conjugate has an apparent circulating half-life of 12 hours, which reflects a composite of both the rate of renal elimination (half-life ∼18 hours) and release of Exa (half-life ∼40 hours). Remarkably, a single low dose of 10 µmol/kg PEG-Exa-only approximately 0.2 µmol/mouse-caused complete suppression of tumor growth of BRCA1-deficient MX-1 xenografts lasting over 40 days. A single low dose of 2.5 µmol/kg PEG-Exa administered with low but efficacious doses of the PARP inhibitor talazoparib showed strong synergy and caused significant tumor regression. Furthermore, the same low, single dose of PEG-Exa administered with the ATR inhibitor VX970 at doses of the DNA damage response inhibitor that do not affect tumor growth show high tumor regression, strong synergy, and synthetic lethality. Significance: A circulating conjugate that slowly releases Exa is described. It is efficacious after a single dose and synergistic with ATR and PARP inhibitors.

A long-acting C-natriuretic peptide for achondroplasia.

The C-natriuretic peptide (CNP) analog vosoritide has recently been approved for treatment of achondroplasia in children. However, the regimen requires daily subcutaneous injections in pediatric patients over multiple years. The present work sought to develop a long-acting CNP that would provide efficacy equal to or greater than that of vosoritide but require less frequent injections. We used a technology for half-life extension, whereby a drug is attached to tetra-polyethylene glycol hydrogels (tetra-PEG) by ß-eliminative linkers that cleave at predetermined rates. These hydrogels-fabricated as uniform ∼60-µm microspheres-are injected subcutaneously, where they serve as a stationary depot to slowly release the drug into the systemic circulation. We prepared a highly active, stable CNP analog-[Gln6,14]CNP-38-composed of the 38 C-terminal amino acids of human CNP-53 containing Asn to Gln substitutions to preclude degradative deamidation. Two microsphere [Gln6,14]CNP-38 conjugates were prepared, with release rates designed to allow once-weekly and once-monthly administration. After subcutaneous injection of the conjugates in mice, [Gln6,14]CNP-38 was slowly released into the systemic circulation and showed biphasic elimination pharmacokinetics with terminal half-lives of ∼200 and ∼600 h. Both preparations increased growth of mice comparable to or exceeding that produced by daily vosoritide. Simulations of the pharmacokinetics in humans indicated that plasma [Gln6,14]CNP-38 levels should be maintained within a therapeutic window over weekly, biweekly, and likely, monthly dosing intervals. Compared with vosoritide, which requires ∼30 injections per month, microsphere [Gln6,14]CNP-38 conjugates-especially the biweekly and monthly dosing-could provide an alternative that would be well accepted by physicians, patients, and patient caregivers.

A very long-acting IL-15: implications for the immunotherapy of cancer.

BACKGROUND: Interleukin-15 (IL-15) is an important cytokine necessary for proliferation and maintenance of natural killer (NK) and CD8+ T cells, and with great promise as an immuno-oncology therapeutic. However, IL-15 has a very short half-life and a single administration does not provide the sustained exposure required for optimal stimulation of target immune cells. The purpose of this work was to develop a very long-acting prodrug that would maintain IL-15 within a narrow therapeutic window for long periods-similar to a continuous infusion. METHODS: We prepared and characterized hydrogel microspheres (MS) covalently attached to IL-15 (MS~IL-15) by a releasable linker. The pharmacokinetics and pharmacodynamics of MS~IL-15 were determined in C57BL/6J mice. The antitumor activity of MS~IL-15 as a single agent, and in combination with a suitable therapeutic antibody, was tested in a CD8+ T cell-driven bilateral transgenic adenocarcinoma mouse prostate (TRAMP)-C2 model of prostatic cancer and a NK cell-driven mouse xenograft model of human ATL (MET-1) murine model of adult T-cell leukemia. RESULTS: On subcutaneous administration to mice, the cytokine released from the depot maintained a long half-life of about 168 hours over the first 5 days, followed by an abrupt decrease to about ~30 hours in accordance with the development of a cytokine sink. A single injection of MS~IL-15 caused remarkably prolonged expansions of NK and ɣδ T cells for 2 weeks, and CD44hiCD8+ T cells for 4 weeks. In the NK cell-driven MET-1 murine model of adult T-cell leukemia, single-agent MS~IL-1550 µg or anti-CCR4 provided modest increases in survival, but a combination-through antibody-depedent cellular cytotoxicity (ADCC)-significantly extended survival. In a CD8+ T cell-driven bilateral TRAMP-C2 model of prostatic cancer, single agent subcutaneous MS~IL-15 or unilateral intratumoral agonistic anti-CD40 showed modest growth inhibition, but the combination exhibited potent, prolonged bilateral antitumor activity. CONCLUSIONS: Our results show MS~IL-15 provides a very long-acting IL-15 with low Cmax that elicits prolonged expansion of target immune cells and high anticancer activity, especially when administered in combination with a suitable immuno-oncology agent.

PET Imaging of the EPR Effect in Tumor Xenografts Using Small 15 nm Diameter Polyethylene Glycols Labeled with Zirconium-89.

The goal was to develop and characterize a companion diagnostic for the releasable PEG40kDa∼SN-38 oncology drug, PLX038, that would identify tumors susceptible to high accumulation of PLX038. PEG conjugates of the zirconium ligand desferroxamine B (DFB) of similar size and charge to PLX038 were prepared that contained one or four DFB, as well as one that contained three SN-38 moieties and one DFB. Uptake and associated kinetic parameters of the 89Zr-labeled nanocarriers were determined in tumor and normal tissues in mice using µPET/CT imaging. The data were fit to physiologically based pharmacokinetic models to simulate the mass-time profiles of distribution of conjugates in the tissues of interest. The time-activity curves for normal tissues showed high levels at the earliest time of measurement due to vascularization, followed by a monophasic loss. In tumors, levels were initially lower than in normal tissues but increased to 9% to 14% of injected dose over several days. The efflux half-life in tumors was very long, approximately 400 hours, and tumor levels remained at about 10% injected dose 9 days after injection. Compared with diagnostic liposomes, the PEG nanocarriers have a longer serum half-life, are retained in tumors at higher levels, remain there longer, and afford higher tumor exposure. The small PEG40kDa nanocarriers studied here show properties for passive targeting of tumors that are superior than most nanoparticles and might be effective probes to identify tumors susceptible to similar size therapeutic nanocarriers such as PLX038.

PLX038: a PEGylated prodrug of SN-38 independent of UGT1A1 activity.

PURPOSE: The purpose of this study was to determine the importance of UGT1A1 activity on the metabolism and pharmacokinetics of a releasable PEG ~ SN-38 conjugate, PLX038A. Irinotecan (CPT-11) is converted to the topoisomerase 1 inhibitor SN-38 by first-pass hepatic metabolism and is converted to its glucuronide SN-38G by UGT1A1. With diminished UGT1A1 activity, the high liver exposure to SN-38 can cause increased toxicity of CPT-11. In contrast, releasable PEG ~ SN-38 conjugates-such as PLX038-release SN-38 in the vascular compartment, and only low levels of SN-38 are expected to enter the liver by transport through the OATP1B1 transporter. METHODS: We measured CPT-11 and PLX038A metabolites in plasma and bile, and determined pharmacokinetics of PLX038A in UGT1A-deficient and replete rats. RESULTS: Compared to CPT-11, treatment of rats with PLX038A results in very low levels of biliary SN-38 and SN-38G, a low flux through UGT1A, and a low SN-38G/SN-38 ratio in plasma. Further, the pharmacokinetics of plasma PLX038A and SN-38 in rats deficient in UGT1A is unchanged compared to normal rats. CONCLUSIONS: The disposition of PEGylated SN-38 is independent of UGT1A activity in rats, and PLX038 may find utility in full-dose treatment of patients who are UGT1A1*28 homozygotes or have metastatic disease with coincidental or incidental liver dysfunction.

Species-specific optimization of PEG~SN-38 prodrug pharmacokinetics and antitumor effects in a triple-negative BRCA1-deficient xenograft.

PURPOSE: Optimal efficacy of a macromolecular prodrug requires balancing the rate of drug release with the rate of prodrug elimination. Since circulating macromolecules have different elimination rates in different species, a prodrug optimal for one species will likely not be for another. The objectives of this work were (a) to develop an approach to optimize pharmacokinetics of a PEG~SN-38 prodrug in a particular species, (b) to use the approach to predict the pharmacokinetics of various prodrugs of SN-38 in the mouse and human, and (c) to develop a PEG~SN-38 conjugate that is optimized for mouse tumor models. METHODS: We developed models that describe the pharmacokinetics of a drug released from a prodrug by the relationship between the rates of drug release and elimination of the prodrug. We tested the model by varying the release rate of SN-38 from PEG~SN-38 conjugates in the setting of a constant prodrug elimination rate in the mouse. Finally, we tested the antitumor efficacy of a PEG~SN-38 optimized for the mouse. RESULTS: Optimization of a PEG~SN-38 prodrug was achieved by adjusting the rate of SN-38 release such that the ratio of t1/2,ß of released SN-38 to the t1/2 of prodrug elimination was 0.2-0.8. Using this approach, we could rationalize the efficacy of previous PEGylated SN-38 prodrugs in the mouse and human. Finally, a mouse-optimized PEG~SN-38 showed remarkable antitumor activity in BRCA1-deficient MX-1 xenografts; a single dose gave tumor regression, suppression, and shrinkage of massive tumors. CONCLUSIONS: The efficacy of a macromolecular prodrug can be optimized for a given species by balancing the rate of drug release from the carrier with the rate of prodrug elimination.

Subcutaneously Administered Self-Cleaving Hydrogel-Octreotide Conjugates Provide Very Long-Acting Octreotide.

We developed a long-acting drug-delivery system that supports subcutaneous administration of the peptidic somatostatin agonist octreotide-a blockbuster drug used to treat acromegaly and neuroendocrine tumors. The current once-a-month polymer-encapsulated octreotide, Sandostatin LAR, requires a painful intragluteal injection through a large needle by a health-care professional. To overcome such shortcomings, Tetra-PEG hydrogel microspheres were covalently attached to the α-amine of d-Phe(1) or the ε-amine of Lys(5) of octreotide by a self-cleaving ß-eliminative linker; upon subcutaneous injection in the rat using a small-bore needle, octreotide was slowly released. The released drug from the ε-octreotide conjugate showed a remarkably long serum half-life that exceeded two months. The α-octreotide conjugate had a half-life of ∼2 weeks, and showed an excellent correlation of in vitro and in vivo drug release. Pharmacokinetic models indicate these microspheres should support once-weekly to once-monthly self-administered subcutaneous dosing in humans. The hydrogel-octreotide conjugate shows the favorable pharmacokinetics of Sandostatin LAR without its drawbacks.

Hydrogel Drug Delivery System Using Self-Cleaving Covalent Linkers for Once-a-Week Administration of Exenatide.

We have developed a unique long-acting drug-delivery system for the GLP-1 agonist exenatide. The peptide was covalently attached to Tetra-PEG hydrogel microspheres by a cleavable ß-eliminative linker; upon s.c. injection, the exenatide is slowly released at a rate dictated by the linker. A second ß-eliminative linker with a slower cleavage rate was incorporated in polymer cross-links to trigger gel degradation after drug release. The uniform 40 µm microspheres were fabricated using a flow-focusing microfluidic device and in situ polymerization within droplets. The exenatide-laden microspheres were injected subcutaneously into the rat, and serum exenatide measured over a one-month period. Pharmacokinetic analysis showed a t1/2,ß of released exenatide of about 7 days which represents over a 300-fold half-life extension in the rat and exceeds the half-life of any currently approved long-acting GLP-1 agonist. Hydrogel-exenatide conjugates gave an excellent Level A in vitro-in vivo correlation of release rates of the peptide from the gel, and indicated that exenatide release was 3-fold faster in vivo than in vitro. Pharmacokinetic simulations indicate that the hydrogel-exenatide microspheres should support weekly or biweekly subcutaneous dosing in humans. The rare ability to modify in vivo pharmacokinetics by the chemical nature of the linker indicates that an even longer acting exenatide is feasible.

Hydrogel drug delivery system with predictable and tunable drug release and degradation rates.

Many drugs and drug candidates are suboptimal because of short duration of action. For example, peptides and proteins often have serum half-lives of only minutes to hours. One solution to this problem involves conjugation to circulating carriers, such as PEG, that retard kidney filtration and hence increase plasma half-life of the attached drug. We recently reported an approach to half-life extension that uses sets of self-cleaving linkers to attach drugs to macromolecular carriers. The linkers undergo ß-eliminative cleavage to release the native drug with predictable half-lives ranging from a few hours to over 1 y; however, half-life extension becomes limited by the renal elimination rate of the circulating carrier. An approach to overcoming this constraint is to use noncirculating, biodegradable s.c. implants as drug carriers that are stable throughout the duration of drug release. Here, we use ß-eliminative linkers to both tether drugs to and cross-link PEG hydrogels, and demonstrate tunable drug release and hydrogel erosion rates over a very wide range. By using one ß-eliminative linker to tether a drug to the hydrogel, and another ß-eliminative linker with a longer half-life to control polymer degradation, the system can be coordinated to release the drug before the gel undergoes complete erosion. The practical utility is illustrated by a PEG hydrogel-exenatide conjugate that should allow once-a-month administration, and results indicate that the technology may serve as a generic platform for tunable ultralong half-life extension of potent therapeutics.

Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates.

Conjugation to macromolecular carriers is a proven strategy for improving the pharmacokinetics of drugs, with many stable polyethylene glycol conjugates having reached the market. Stable conjugates suffer several limitations: loss of drug potency due to conjugation, confining the drug to the extracellular space, and the requirement for a circulating conjugate. Current research is directed toward overcoming such limitations through releasable conjugates in which the drug is covalently linked to the carrier through a cleavable linker. Satisfactory linkers that provide predictable cleavage rates tunable over a wide time range that are useful for both circulating and noncirculating conjugates are not yet available. We describe such conjugation linkers on the basis of a nonenzymatic ß-elimination reaction with preprogrammed, highly tunable cleavage rates. A set of modular linkers is described that bears a succinimidyl carbonate group for attachment to an amine-containing drug or prodrug, an azido group for conjugation to the carrier, and a tunable modulator that controls the rate of ß-eliminative cleavage. The linkers provide predictable, tunable release rates of ligands from macromolecular conjugates both in vitro and in vivo, with half-lives spanning from a range of hours to >1 y at physiological pH. A circulating PEG conjugate achieved a 56-fold half-life extension of the 39-aa peptide exenatide in rats, and a noncirculating s.c. hydrogel conjugate achieved a 150-fold extension. Using slow-cleaving linkers, the latter may provide a generic format for once-a-month dosage forms of potent drugs. The releasable linkers provide additional benefits that include lowering C(max) and pharmacokinetic coordination of drug combinations.

Molecular modeling and crystal structure of ERK2-hypothemycin complexes.

Resorcylic acid lactones containing a cis-enone-such as hypothemycin-are susceptible to Michael addition reactions and are potent and specific inhibitors of about 45 of the known Ser/Thr/Tyr protein kinases. These inhibitors bind reversibly, and then form a covalent adduct with a completely conserved cysteine in the ATP binding site of their target kinases. As a paradigm for the structures of the cis-enone resorcylic acid lactone complexes with this subset of kinases, we have modeled the structure of ERK2-hypothemycin reversible and covalent complexes using docking and extended molecular dynamics simulations. Subsequently, we determined the 2.5A resolution crystal structure of the complex that was in excellent accord with the modeled structure. The results were used to discuss structure-activity relationships, and provide a structural template for the development of irreversible inhibitors that complement the ATP binding site of kinases.

Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides.

Resorcylic acid lactones containing a cis-enone are susceptible to Michael addition reactions and are potent inhibitors of several protein kinases. A structural-bioinformatics analysis identified a conserved Cys residue in the ATP-binding site of the kinases reported to be inhibited by cis-enone resorcylic acid lactones but absent in those that are not. Mining of the kinome database revealed that a subset of some 46 kinases contained this Cys residue. Screening a panel of 124 kinases with the resorcylic acid lactone hypothemycin showed that 18 of 19 targets containing the conserved Cys were inhibited. Kinetic analyses showed time-dependent inhibition, a hallmark of covalent inactivation, and biochemical studies of the interaction of extracellular signal-regulated kinase (ERK)2 with hypothemycin confirmed covalent adduct formation. Resorcylic acid lactones are unique among kinase inhibitors in that they target mitogen-activated protein (MAP) kinase pathways at four levels: mitogen receptors, MAP kinase kinase (MEK)1/2 and ERK1/2, and certain downstream ERK substrates. Cell lines dependent on the activation of Tyr kinase mitogen receptor targets of the resorcylic acid lactones were unusually sensitive toward hypothemycin and showed the expected inhibition of kinase phosphorylation due to inhibition of the mitogen receptors and/or MEK1/2 and ERK1/2. Among cells without mitogen receptor targets, those harboring an ERK pathway-activating B-RAF V600E mutation were selectively and potently inhibited by hypothemycin. Hypothemycin also prevented stimulated activation of the p38 cascade through inhibition of the Cys-containing targets MEK3/6 and TGF-beta-activated kinase 1 and of the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) cascade through inhibition of MEK4/7.

The biosynthetic genes for disorazoles, potent cytotoxic compounds that disrupt microtubule formation.

Disorazoles are polyketides produced by the myxobacterium Sorangium cellulosum So ce12. Their mode of action is to inhibit tubulin polymerization and destabilize microtubules. Using transposon mutagenesis, two mutant strains were identified that produced no disorazoles. Sequencing the DNA flanking the insertions revealed a polyketide synthase gene cluster that would encode three polypeptides, DszA, DszB, and DszC, with DszC containing both nonribosomal peptide synthetase and polyketide synthase modules. The disorazole polyketide synthase modules lack an acyltransferase domain. Instead, a separate gene, dszD, encodes an AT protein, thus revealing that the disorazole gene cluster falls into the trans-AT Type I family of PKS enzymes.

Insights into the biosynthesis of the benzoquinone ansamycins geldanamycin and herbimycin, obtained by gene sequencing and disruption.

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.

The leptomycin gene cluster and its heterologous expression in Streptomyces lividans.

Leptomycin exerts its antifungal and anti-tumoral activity via inhibiting nucleo-cytoplasmic translocations in eukaryotic cells. To learn more about the biosynthesis of leptomycin and in an effort to generate leptomycin analogues through genetic engineering, 90 kb segment of DNA containing the putative leptomycin (lep) biosynthesis cluster from Streptomyces sp. ATCC 39366 was cloned and sequenced. The lep cluster consist of 12 polyketide synthase (PKS) modules distributed in four genes (lepA, B, C and D) and a P450 encoding gene. The lep gene cluster was confirmed by its successful expression in Streptomyces lividans, where it directed the production of the two natural congeners-leptomycins A and B. The production of leptomycin B showed that the host has the capability to synthesize ethylmalonyl-CoA.