Manoj K. Mishra

Dendrimer-based targeted intravitreal therapy for sustained attenuation of neuroinflammation in retinal degeneration.

Retinal neuroinflammation, mediated by activated microglia, plays a key role in the pathogenesis of photoreceptor and retinal pigment epithelial cell loss in age-related macular degeneration and retinitis pigmentosa. Targeted drug therapy for attenuation of neuroinflammation in the retina was explored using hydroxyl-terminated polyamidoamine (PAMAM) dendrimer-drug conjugate nanodevices. We show that, upon intravitreal administration, PAMAM dendrimers selectively localize within activated outer retinal microglia in two rat models of retinal degeneration, but not in the retina of healthy controls. This pathology-dependent biodistribution was exploited for drug delivery, by covalently conjugating fluocinolone acetonide to the dendrimer. The conjugate released the drug in a sustained manner over 90 days. In vivo efficacy was assessed using the Royal College of Surgeons (RCS) rat retinal degeneration model over a four-week period when peak retinal degeneration occurs. One intravitreal injection of 1 μg of FA conjugated to 7 μg of the dendrimer was able to arrest retinal degeneration, preserve photoreceptor outer nuclear cell counts, and attenuate activated microglia, for an entire month. These studies suggest that PAMAM dendrimers (with no targeting ligands) have an intrinsic ability to selectively localize in activated microglia, and can deliver drugs inside these cells for a sustained period for the treatment of retinal neuroinflammation.

Drug release characteristics of PAMAM dendrimer-drug conjugates with different linkers.

Drug release from polymer-drug conjugates plays a crucial role on the efficacy. This is especially true for dendrimers where there is a steric crowding at the surface. The drug release characteristics of G4-polyamidoamine (PAMAM) dendrimer-ibuprofen conjugates with ester, amide, and peptide linkers were investigated, in addition to a linear PEG-ibuprofen conjugate to understand the effect of architecture and linker on drug release. Ibuprofen was directly conjugated to NH(2)-terminated dendrimer by an amide bond and OH-terminated dendrimer by an ester bond. A tetra-peptide-linked dendrimer conjugate and a linear mPEG-ibuprofen conjugate were also studied for comparison to direct linked dendrimer conjugates. Amide-linked conjugates were relatively stable against hydrolysis, whereas the ester-linked conjugates showed pH-dependent release and the extent of release varied with pH from 3% (pH 5) to 38% (pH 8.5) for the 10-day period studied. Direct amide- and ester-linked conjugates did not release ibuprofen enzymatically in cathepsin B buffer and diluted human plasma. In contrast, mPEG conjugate released 65% of its payload within 12 h in diluted plasma by esterase activity, and the peptide-linked dendrimer conjugate released 40% of its payload within 48 h by cathepsin B activity. It is demonstrated that the steric crowding at the surface of PAMAM dendrimer-drug conjugates, along with linking chemistry govern the drug release mechanisms as well as kinetics. Understanding these structural and steric effects on their drug release characteristics is crucial for the design of dendrimer conjugates with high efficacy.

Intrinsic targeting of inflammatory cells in the brain by polyamidoamine dendrimers upon subarachnoid administration

AIM Understanding the interactions between nanomaterials and disease processes is crucial for designing effective therapeutic approaches. This article explores the unusual neuroinflammation targeting of dendrimers (with no targeting ligands) in the brain, with significant consequences for nanoscale materials in medicine. METHOD The in vivo biodistribution of fluorescent-labeled neutral generation-4- polyamidoamine dendrimers (∼4 nm) in a rabbit model of cerebral palsy was explored following subarachnoid administration. RESULTS These dendrimers, with no targeting ligands, were localizing in activated microglia and astrocytes (cells responsible for neuroinflammation), even in regions far moved from the site of injection, in newborn rabbits with maternal inflammation-induced cerebral palsy. CONCLUSION This intrinsic ability of dendrimers to localize inactivated microglia and astrocytes can enable targeted delivery of therapeutics in disorders such as cerebral palsy, Alzheimers and multiple sclerosis.

PAMAM dendrimer-azithromycin conjugate nanodevices for the treatment of Chlamydia trachomatis infections

UNLABELLED Chlamydia trachomatis is an important bacterial pathogen known to be etiological in genital infections, as well as several serious disease sequelae, including inflammatory arthritis. Chlamydiae can persist in infection, making treatment with antibiotics such as azithromycin (AZ) a challenge. The authors explore the use of neutral generation-4 polyamidoamine (PAMAM) dendrimers as intracellular drug-delivery vehicles into chlamydial inclusions. Azithromycin was successfully conjugated with the dendrimers, and the conjugate (D-AZ) released ≈ 90% of the drug over 16 hours. The conjugate readily entered both the Chlamydia-infected HEp-2 cells and the chlamydial inclusions. The conjugate was significantly better than free drug in preventing productive infections in the cells when added at the time of infection, and better in reducing the size and number of inclusions when added either 24 hours or 48 hours post infection. These studies show that dendrimers can deliver drugs efficiently to growing intracellular C. trachomatis, even if the organism is in the persistent form. FROM THE CLINICAL EDITOR In this report, the use of polyamidoamine dendrimers as intracellular drug-delivery vehicles into chlamydial inclusions is investigated. This method results in efficient intracellular delivery of azithromycin to address chlamydia infection.

Interaction of cocaine-, benztropine-, and GBR12909-like compounds with wild-type and mutant human dopamine transporters: molecular features that differentially determine antagonist-binding properties

The widely abused psychostimulant cocaine is thought to elicit its reinforcing effects primarily via inhibition of the neuronal dopamine transporter (DAT). However, not all DAT inhibitors share cocaine’s behavioral profile, despite similar or greater affinity for the DAT. This may be due to differential molecular interactions with the DAT. Our previous work using transporter mutants with altered conformational equilibrium (W84L and D313N) indicated that benztropine and GBR12909 interact with the DAT in a different manner than cocaine. Here, we expand upon these previous findings, studying a number of structurally different DAT inhibitors for their ability to inhibit [3H]CFT binding to wild‐type, W84L and D313N transporters. We systematically tested structural intermediates between cocaine and benztropine, structural hybrids of benztropine and GBR12909 and a number of other structurally heterologous inhibitors. Derivatives of the stimulant desoxypipradrol (2‐benzhydrylpiperidine) exhibited a cocaine‐like binding profile with respect to mutation, whereas compounds possessing the diphenylmethoxy moiety of benztropine and GBR12909 were dissimilar to cocaine‐like compounds. In tests with specific isomers of cocaine and tropane analogues, compounds with 3α stereochemistry tended to exhibit benztropine‐like binding, whereas those with 3β stereochemistry were more cocaine‐like. Our results point to the importance of specific molecular features – most notably the presence of a diphenylmethoxy moiety – in determining a compound’s binding profile. This study furthers the concept of using DAT mutants to differentiate cocaine‐like inhibitors from atypical inhibitors in vitro. Further studies of the molecular features that define inhibitor–transporter interaction could lead to the development of DAT inhibitors with differential clinical utility.

Dendrimer-enabled modulation of gene expression in Chlamydia trachomatis.

The obligate intracellular bacterium Chlamydia trachomatis is an important human pathogen. The genome of this organism is small but encodes many genes of currently unknown function that are thought to be involved in virulence. Lack of a system for genetic manipulation has been a key challenge to advancing the understanding of molecular genetics underlying virulence for this bacterium. We developed a dendrimer-enabled system for transformation of C. trachomatis, and used it to demonstrate the efficient and highly specific knockdown of transcript levels from targeted genes. Antisense, sense, and other control oligonucleotides targeting two sets of duplicated genes on the chlamydial chromosome were designed, commercially synthesized, and complexed with generation-4 polyamidoamine (PAMAM) dendrimers. The complexes were given to HEp-2 cell cultures infected for 16 h with C. trachomatis serovar K and then removed three hours later. Infected cultures were harvested 6 h after pulsing, and DNA and RNA/cDNA were prepared for assessment of transcript levels compared to those for the same genes in infected cultures, without dendrimer complexation. In all cases, the targeted gene complexed to dendrimer, but not its duplicate, showed up to 90% transcript attenuation. The duration of attenuation can be extended by repeated pulsing, and in some cases transcript levels from multiple genes can be attenuated in the same organism. This system will allow study of chlamydial gene function in pathogenesis, leading to more effective therapies to treat Chlamydia-induced diseases in a targeted manner.

Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells

Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (∼ 21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ∼ 100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications.

A Dendrimer-based Immunosensor for Improved Capture and Detection of Tumor Necrosis Factor-α Cytokine

A dendrimer-based sandwich type enzyme-linked immunosorbent assay (ELISA) was developed for the improved detection of recombinant human tumor necrosis factor-alpha (TNF-α) for early diagnosis of perinatal diseases. Hydroxyl-terminated generation four poly(amidoamine) dendrimer (G4-OH) was used for the development of a solid phase bio-sensing platform. The surface of the ELISA plate was modified with polyethylene-glycol (PEG) and thiol-functionalized G4-OH was immobilized on the PEG-functionalized plate. A capture antibody was oxidized and covalently immobilized onto the dendrimer-modified ELISA plate, which provides favorable orientation for the antigen binding sites toward the analyte. The dendrimer-modified plate showed enhanced sensitivity, and the detection limit for TNF-α was found to be 0.48 pg mL(-1), which is significantly better than the commercially available ELISA kit. The selectivity of the dendrimer-modified ELISA plate was further evaluated with a mixture of cytokines, which showed results for similar to that of TNF-α alone. The modified plate provides a greater opportunity for the detection of a wide range of cytokines and biomarkers.

Investigation of various N-heterocyclic substituted piperazine versions of 5/ 7-{[2-(4-Aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol: Effect on affinity and selectivity for dopamine D3 receptor

Here we report on the design and synthesis of several heterocyclic analogues belonging to the 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol series of molecules. Compounds were subjected to [(3)H]spiperone binding assays, carried out with HEK-293 cells expressing either D2 or D3 dopamine receptors, in order to evaluate their inhibition constant (K(i)) at these receptors. Results indicate that N-substitution on the piperazine ring can accommodate various substituted indole rings. The results also show that in order to maintain high affinity and selectivity for the D3 receptor the heterocyclic ring does not need to be connected directly to the piperazine ring as the majority of compounds included here are linked either via an amide or a methylene linker to the heterocyclic moiety. The enantiomers of the most potent racemic compound 10e exhibited differential activity with (-)-10e (K(i); D2=47.5 nM, D3=0.57 nM) displaying higher affinity at both D2 and D3 receptors compared to its enantiomer (+)-10e (K(i); D2=113 nM, D3=3.73 nM). Additionally, compound (-)-10e was more potent and selective for the D3 receptor compared to either 7-OH-DPAT or 5-OH-DPAT. Among the bioisosteric derivatives, the indazole derivative 10g and benzo[b]thiophene derivative 10i exhibited the highest affinity for D2 and D3 receptors. In the functional GTPgammaS binding study, one of the lead molecules, (-)-15, exhibited potent agonist activity at both D2 and D3 receptors with preferential affinity at D3.

Further structural optimization of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine and 1,4-diazabicyclo[3.3.1]nonane derivatives by introducing an exocyclic hydroxyl group: interaction with dopamine, serotonin, and norepinephrine transporters.

Our earlier effort to develop constrained analogues of flexible piperidine derivatives for monoamine transporters led to the development of a series of 3,6-disubstituted piperidine derivatives, and a series of 4,8-disubstituted 1,4-diazabicyclo[3.3.1]nonane derivatives. In further structure-activity relationship (SAR) studies on these constrained derivatives, several novel analogues were developed where an exocyclic hydroxyl group was introduced on the N-alkyl-aryl side chain. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin (5-HT) transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. Compounds were also tested for their binding potency at the DAT by their ability to inhibit binding of [(3)H]WIN 35,428. The results indicated that position of the hydroxyl group on the N-alkyl side chain is important along with the length of the side chain. In general, hydroxyl derivatives derived from more constrained bicyclic diamines exhibited greater selectivity for interaction with DAT compared to the corresponding 3,6-disubstituted diamines. In the current series of molecules, compound 11b with N-propyl side chain with the hydroxyl group attached in the benzylic position was the most potent and selective for DAT (K(i)=8.63nM; SERT/DAT=172 and NET/DAT=48.4).