Stampidine: a selective oculo-genital microbicide

Osmond J. D'Cruz1,2,* and Fatih M. Uckun1

1 Parker Hughes Institute, 2657 Patton Road, St Paul, MN 55113, USA; 2 Paradigm Pharmaceuticals, LLC, St Paul, MN, USA


* Corresponding author. Tel: +1-651-628-9988; Fax: +1-651-628-9891; E-mail: odcruz{at}ih.org


    Abstract
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
Adenoviruses (ADVs) are causative agents of severe and extremely contagious ocular and genital infections associated with conjunctivitis, genital ulcers and urethritis. Yet, no functional antiviral compounds are currently available against adenoviral infections. We discovered halogen-substituted phenyl phosphoramidate derivatives of stavudine (STV/d4T) as a new class of dual-function anti-human immunodeficiency virus (HIV) agents with potent and selective anti-ADV activity. The lead compound, stampidine [5'-(4-bromophenyl methoxyalaninylphosphate)-2',3'-didehydro-3'-deoxythymidine], was the most potent non-toxic dual-function antiviral agent. Stampidine displayed remarkable in vitro and in vivo anti-HIV activity against drug-sensitive and drug-resistant HIV strains. Stampidine was non-cytotoxic and nonirritating to mucosal epithelial cells. Several preclinical studies conducted thus far, suggest that stampidine has clinical potential as a dual-function topical agent for the prevention and/or effective treatment of oculo-genital ADV/HIV infections.

Keywords: adenovirus , conjunctivitis , contact lens , HIV/AIDS , eye infections , stavudine , d4T


    Introduction
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
Nosocomial virus infections of the eye such as viral conjunctivitis caused mainly by adenoviruses (ADVs) are a highly contagious eye disease that occurs worldwide.15 Epidemic keratoconjunctivitis (EKC), pharyngo-conjunctival fever, follicular conjunctivitis, and chronic papillary conjunctivitis are four examples of adenoviral ocular infections that are associated with community and medical facility epidemics.18 Ocular ADV infections can be acquired by swimming in contaminated water, towels, ophthalmic solutions, ophthalmic instruments, or fingers.916 In fact, ADV can be recovered from ophthalmic solutions, non-porous surfaces, and hands up to 49 days after inoculation.1719 Because the number of people wearing contact lenses has swelled to 90 million worldwide—35 million nationwide,20 the most serious complication of extended contact lens wear is sight threatening corneal ulceration from keratitis.21 Contaminated contact lenses may serve as a reservoir for spread of adenoviral infection to family, friends and co-workers.22 Although not permanently blinding, ocular adenoviral infections are associated with significant patient morbidity, including symptomatic distress, with visual disturbances that can last months to years.1,23


    Ocular and genital ADV serotypes
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
The ADV family consists of 51 known serotypes, which fall into six subgroups (A to F) based on oncogenicity, haemagglutination patterns and DNA homology.2426 About one-half of these serotypes can cause ocular infections worldwide. Thirty-two serotypes constitute subgroup D that are significantly associated with oculo-genital infections.1,3,2325 Subgroup B, C, D and E adenoviruses have been isolated from conjunctivitis (viral pink eye) patients,2729 while EKC which mainly affects the cornea, occurs most commonly with infection by subgroup D adenoviruses of serotype 8, 19 or 37.1,4,11,16,17,30,31 Adenovirus 8 is a common cause of EKC that may be transmitted by ocular examination apparatus.11,16,32 Occasionally, serotypes 9, 15 and 22 have also been reported as a cause.30,32 Acute keratoconjunctivitis with prominent subconjunctival haemorrhage has been associated with serotypes 19 and 37.33 Serotypes 9, 15, 17 and 28 are known to induce relatively mild follicular conjunctivitis while serotype 34 induces acute follicular conjunctivitis.7,34 Serotypes 3, 4 and 7, which belong to other subgroups, also cause conjunctivitis and pharyngo-conjunctival fever.35,36

ADV serotypes 2, 8, 19 and 37 have been isolated from the genital tract of patients attending sexually transmitted disease clinics.3741 Genital ADV infections are associated with urethritis and penile ulcers in men,39,40 and cervicitis and labial ulcers in women3739,42 (Table 1). Some patients with genital ADV infection also have conjunctivitis caused by the same serotype.3844 Additionally, ADVs are associated with a significant number of human upper respiratory (B, C and E), gastrointestinal (A and F), and urinary tract (B) infections. The clinical manifestations of these ADV infections range from a symptomatic excretion of the virus to disseminated disease with multiorgan failure and death, especially in patients with immunological deficiencies.4552 Many HIV-infected patients with AIDS shed ADVs that are rarely or never isolated from immunocompetent individuals.4551 Typical AIDS-associated ADV serotypes include types 9, 17, 20, 22, 23, 26, 27 and 42–51.45,47,51 Adenovirus subtypes 2/5 are also being used as vectors for gene delivery in clinical trials to treat a variety of acquired and inherited diseases.5357


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Table 1. Oculo-genital tropism of group D adenoviral serotypes

 

    Ocular and genital ADV receptors
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 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
Oculo-genital ADVs use receptors with specific tropisms.44 The ADV fibre protein is responsible for attachment of the virus to cell surface receptors.34,43,44 Fibres of serotype 19 and 37 are identical suggesting that ADV 19 and 37 tropism for the eye and genital tract is mediated by the expression of a common receptor.43,44 Adenoviruses (8, 19 and 37) that cause oculo-genital infections use either sialic acid linked to galactose via {alpha}2,3 glycosidic bonds or the complement membrane cofactor protein (CD46) as cellular receptors.43,5860 The major sialylated components of ocular and genital environments are the gel forming and membrane-bound mucins rich in {alpha}2,3-linked sialic acid (MUC1-17).6163 Adenoviruses that use CD46 also cause cystitis that may be lethal to immunocompromised patients.48 Members of the same ADV family may use distinct modes of interaction to bind to the same receptor and members of the same ADV subgroup may use different receptors.60 CD46 is selectively expressed in corneal and genital epithelia and in ocular as well as genital tract fluids.6467 The tropism of ADV serotypes 8, 19 and 37 is thought to be due to their preferential use of sialic acid linkage as well as to certain CD46 isoforms that are abundantly expressed on cells and secretions of the eye and genital tract.


    Ocular manifestations of ADV infection
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 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
The cornea is normally considered a ‘privileged’ site because of its avascularity and lack of lymphatic vessels.68 Antigens, cytokines, inflammatory mediators and leucocytes that enter into the cornea must do so from the limbic and/or ciliary body vessels. Inflammatory cytokines and/or chemotactic gradients that are elicited locally by corneal cells can profoundly affect the emigration of leucocytes from the limbic and ciliary circulation to the cornea.6870 Keratocytes, the resident cells within the corneal stroma, maintain the corneal extracellular matrix in a precisely organized and transparent state. Keratocytes amplify corneal stromal inflammatory responses to chemical and infectious stimuli leading to corneal injury and invasion. The corneal manifestations of EKC include epithelial keratitis, macro-epithelial erosions, and delayed-onset, multifocal, subepithelial infiltrates.6972 Subepithelial stromal infiltrates follow ADV infection of corneal epithelium, secondary viral antigen deposition in the superficial corneal stroma, and lymphocyte infiltration in a multifocal pattern at sites of antigen–antibody complexes resulting in up-regulation of inflammatory cell chemotactants by corneal stromal cells. The corneal infiltrates of EKC cause significant ocular morbidity; reduced vision, photophobia, and foreign body sensation which may persist for months to years after infection.68 Irreversible damage to the eye can occur in cases of fulminant inflammation. Clearly, the desirable outcome is to control the infection resulting in re-epithelialization and healing with minimum damage to vision. Consequently, identifying new agents for the prevention of ADV infections is of extreme importance in preventing corneal damage related to infection, inflammation and physical wounding.


    Limitations of current anti-ADV agents
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
Despite significant knowledge of the molecular biology and genetics of ADV, currently there is no clinically effective antiviral agent for the prevention or treatment of ADV infections. Several nucleoside analogues, including 2,3-dideoxynucleoside 5-triphosphates [2'3'-dideoxycytidine triphosphate (TP), 2',3'-dideoxyadenosine TP and 2',3'-dideoxythymidine TP], 3-fluorosubstituted nucleoside TP inhibitors (3'-fluorothymidine TP, 2',3'-dideoxy-3'-fluoroguanosine TP and 2',3'-dideoxy-3'-fluorouridine TP) and 3-fluoro-2-deoxythymidine, have been discovered as having antiviral activity against ADV by inhibiting the viral DNA polymerase-mediated DNA replication in ADV-infected cells.7376 Most nucleoside analogues exhibit broad-spectrum antiviral activity.77,78 Nucleoside analogues require activation (phosphorylation) by cellular enzymes to exert their antiviral activity. The active intracellular diphosphate form of the drug exerts its mechanism of action as both a competitive inhibitor and an alternative substrate for 2-deoxycytidine 5'-TP in the viral DNA polymerase reaction.

Ribavirin (Virazole®; 1-ß-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a guanosine analogue that inhibits the replication of many RNA and DNA viruses.77 The antiviral mechanism of ribavirin is due to inhibition of viral encoded polymerases, inhibition of genomic RNA capping, or inhibition of cellular encoded enzymes that control de novo synthesis of purine nucleosides. Cidofovir [HPMPC, Vistide, (S)-1-(3-hydroxy-2-phosphonomethoxypropyl)cytosine dihydrate] is a nucleotide analogue that contains a phosphonate group that does not require cellular phosphorylation. Both ribavirin and cidofovir exhibit inhibitory activity in vitro against several RNA and DNA viruses (adeno-, arena-, bunya-, cytomegalo-, Epstein–Barr-, hepadna-, herpes-, irido-, myxo-, papilloma-, paramyxo-, polyoma-, pox-, retroviruses or RNA tumour viruses).7780 Cellular enzymes convert cidofovir into the active diphosphate form. Cidofovir is currently being pursued in the topical/intravenous/intravesicular treatment of ADV infections.78,80,81 The momentum in the design of novel nucleoside analogues for anti-ADV therapy is their ability to discriminate between the viral DNA polymerase and cellular DNA polymerases. None of the currently available anti-HIV agents has potent anti-ADV activity and several experimental anti-ADV agents are associated with significant side effects. Development of nephrotoxicity is the major risk factor encountered with patients receiving cidofovir, whereas extravascular haemolysis, bone marrow suppression, and anaemia are the most common dose-limiting toxic effects seen with ribavirin.78,8184 The local toxicity of cidofovir to the skin of the eyelids and the conjunctiva is clinically similar to the signs of initial ADV inflammation. Consequently, there is an urgent need for selective anti-ADV agents with more favourable safety profiles than the available nucleoside and nucleotide analogues as well as dual-function anti-HIV agents with anti-ADV activity.


    Stampidine, a novel anti-ADV agent
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
Stavudine (STV)/d4T is a pyrimidine nucleoside analogue used in the treatment of HIV infection.85 It inhibits viral reverse transcriptase (RT) as do zidovudine (ZDV)/AZT, didanosine (ddI), zalcitabine (ddC) and lamivudine (3TC), which comprise the family of nucleoside RT inhibitors (NRTIs).8486 The 5-triphosphates of these NRTIs, which are generated intracellularly by the action of nucleoside and nucleotide kinases, are potent inhibitors of HIV-1 RT.87,88 The rate-limiting step for the intracellular generation of the bioactive STV metabolite STV-TP is the conversion of STV into its monophosphate (MP) derivative.87 In an attempt to overcome the dependence of STV on intracellular nucleoside kinase activation, we prepared novel aryl phosphate derivatives of STV and evaluated their broad-spectrum antiviral activity. These prodrugs have been shown to undergo intracellular hydrolysis to yield MP derivatives independently of thymidine kinase (TK) and are further phosphorylated by TK to give the bioactive TP derivatives.8790 Halogen-substituted phenyl phosphoramidate derivatives of STV were identified as a new class of dual-function anti-HIV agents with potent and selective anti-ADV activity.91,92

Table 2 lists the anti-ADV activity of 12 structurally similar STV derivatives. All 12 derivatives of STV were substantially more potent than STV and inhibited ADV-induced plaque formation at nanomolar IC50 values. Compounds with an electron-withdrawing group (chloro, bromo or di-halogen) at the para position of the phenyl group, which led to enhanced rates of hydrolysis to yield Ala-STV-MP, were the most active compounds. In contrast, electron-donating substituents such as methoxy or methyl-substituted compounds showed a 5- to 10-fold decrease in activity. The position of the substituents on the phenyl ring did not alter the activity profiles. Cluster analysis of log IC50 values for the ADV strain versus Hammett sigma values and partition coefficient (P) values revealed bivariate plots.92 Compounds with higher log P values were more active. However, lipophilicity plays a minor role in predicting the anti-ADV activity of these phosphoramidate derivatives. Unsubstituted, as well as the electron-donating group substituted phosphoramidate derivatives had the highest solubility in water (12–45 mg/mL).


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Table 2. Dual-function antiviral activity and physicochemical properties of aryl phosphoramidate derivatives of stavudine

 
Stampidine [5'-(4-bromophenyl methoxyalaninylphosphate)-2',3'-didehydro-3'-deoxythymidine] was the least cytotoxic and most effective of the STV derivatives against ADV. An STV derivative with a 4-chloro substitution was equally potent as a dual-function anti-HIV and anti-ADV agent. The lead compound, stampidine inhibited ADV-induced plaque formation in human foreskin fibroblasts (CRL-2097) in a concentration-dependent fashion with a mean IC50 value of 22 nM without any evidence of cytotoxicity even at 100 µM (selectivity index >4000). Unlike the non-specific antiviral agents ribavirin or cidofovir, stampidine was selective against ADV and lacked antiviral activity against human cytomegalovirus, type 1 (oral) or type 2 (genital) herpes simplex viruses, enterovirus ECHO 30, and respiratory syncytial virus92 (Table 3). These results established that halogen-substituted phenyl phosphoramidate derivatives of STV are selective anti-HIV/ADV agents.


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Table 3. Selective antiviral activity of stampidine versus cidofovir and ribavirin against mucosotropic human viruses

 

    Stampidine, a novel broad-spectrum anti-HIV agent
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
Table 4 summarizes the in vitro activity profile of stampidine against HIV-1 isolates with B- and non-B envelope subtypes (A, C, F and G) originating from Asia, South America and sub-Saharan Africa.9395 Stampidine displayed potent anti-HIV activity against the HIV-1 strain HTLVIIIB in human peripheral blood mononuclear cells (PBMCs) with nanomolar IC50 and IC90 values and a selectivity index of >100 000. The mean IC50 value of stampidine against HTLVIIIB was 20-times lower than the mean IC50 value of STV (IC50 values 1 nM versus 18 nM, P < 0.001; Table 2). Similarly, in a side-by-side comparison against 10 ZDV-susceptible primary clinical HIV-1 isolates, nine of which had a non-B envelope subtype, stampidine was 100-fold more potent than STV (IC50 values 2 nM versus 240 nM, P < 0.0001).93 Stampidine was also active against phenotypically and/or genotypically NRTI-resistant HIV and inhibited the replication of 20 ZDV-resistant primary clinical HIV-1 isolates (average ZDV IC50 = 2 µM) with low nanomolar to subnanomolar IC50 values (average IC50 value of 9 nM) (Table 4). Similarly, stampidine inhibited the replication of multiple laboratory HIV-1 strains and primary clinical HIV-1 isolates with non-nucleoside RT inhibitor (NNRTI) binding site mutations (K103N, V106N, Y179I, Y181C, Y188L) and/or phenotypic NNRTI-resistant profile with an average IC50 value of 11 nM94 (Table 4). Stampidine was active against HIV-1 isolates with five thymidine analogue mutations at subnanomolar concentrations.


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Table 4. Broad-spectrum anti-HIV activity of stampidine

 
The in vivo pharmacokinetics, metabolism and toxicity of stampidine have been examined in two rodent (mice and rats) and three non-rodent (cats, rabbits and dogs) species.9699 Stampidine forms two active Phase I metabolites, Ala-STV-MP and STV, with favourable pharmacokinetics (Figure 1). Ala-STV-MP is stable both in plasma and in whole blood (>24 h). Para-bromophenyl sulphate was the major in vivo Phase II metabolite. Intracellular metabolism of stampidine to Ala-STV-MP and STV was mediated by physostigmine-sensitive cholinesterase and paraoxon-sensitive carboxylesterase.



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Figure 1. Chemical structure of stampidine and its major metabolites. Stampidine [2',3'-didehydro-3'-deoxythymidine 5'-(p-bromophenyl methoxyalaninyl phosphate)] differs from stavudine (STV/d4T) by the presence of a phenyl phosphate group with an alanine side chain and a bromo substituent on the C-5 position of the phenyl ring. The prodrug forms two active Phase I intracellular metabolites, namely alaninyl-STV-monophosphate (Ala-STV-MP) and STV [2',3'-didehydro-3'-deoxythymidine 5'-(alaninyl phosphate)]. Para-bromophenyl sulphate is the major in vivo Phase II metabolite.

 
Orally administered non-toxic doses of stampidine demonstrated significant and dose-dependent in vivo anti-HIV activity against the NRTI-resistant clinical HIV-1 isolate BR/92/019 in severe combined immunodeficient (SCID) mice reconstituted with PBMCs from seronegative donors.96 In the feline immunodeficiency virus (FIV)/domestic cat model for AIDS, a single oral bolus dose (50 mg/kg or 100 mg/kg) of stampidine yielded therapeutic concentrations of the prodrug and its active metabolite, Ala-STV-MP, at >4-log higher than their respective IC50 values which was achieved and maintained for more than 4 h after parenteral as well as oral administration without any adverse reactions.97 Stampidine displayed potent antiretroviral activity as evidenced by a ≥1-log decrease in the FIV load of circulating PBMCs.97 Stampidine was very well tolerated in BALB/c and CD-1 mice, Lewis rats, Beagle dogs and domestic cats without any detectable acute or subacute toxicity at single intraperitoneal or oral bolus dose levels as high as 600 mg/kg (Table 5). Daily intraperitoneal or oral administration of stampidine for up to eight consecutive weeks was not associated with any detectable acute, subacute, or chronic toxicity at cumulative dose levels as high as 8.4 g/kg (Table 5).9499


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Table 5. Summary of stampidine safety and efficacy

 
The documented in vitro potency of stampidine against primary clinical HIV-1 isolates with genotypic and/or phenotypic NRTI- or NNRTI-resistance as well as non-B envelope subtype together with its in vivo antiretroviral activity in HIV-infected human-PBMC-SCID mice as well as FIV-infected cats prompted us to further develop this dual-function agent for clinical use.


    Solubility and formulation studies
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
In addition to its potent dual-function properties, stampidine has been evaluated for stability, formulation, bioavailability and mucosal toxicity.100109

Stability

Based on accelerated stability studies conducted at 40°C, stampidine is a stable molecule at room temperature for more than 6 months. A comparison of the hydrolytic and biological activities of dual-function STV derivatives revealed that the introduction of electron-withdrawing substituents enhanced the antiviral activity of these compounds and the rate of hydrolysis predicted the potency of the compounds.91,92,95,109 All compounds were efficacious against HIV-1 (1–25 nM). Regression analysis revealed a significant linear relationship between anti-HIV (A17) IC50 values and Hammett sigma values (P = 0.036) or log 10 transformed hydrolysis rates (P = 0.014).92 However, these aryl phosphate derivatives differed more than 60-fold in the rates of alkaline hydrolysis.92,103 The presence of electron-withdrawing groups at the phenyl moiety enhanced the rate of hydrolysis of these phosphoramidate derivatives to yield Ala-STV-MP.91,109

In various tissue microenvironments, the metabolism of stampidine may occur through the action of hydrolytic enzymes. Accordingly, we demonstrated that stampidine and other halogen-substituted phosphoramidate derivatives of STV can be activated by esterase, lipase or protease-mediated hydrolysis.101103 The target site for the lipase appears to be the methyl ester group of the L-alanine side chain. As a result of the stereochemistry of its phosphorous chiral centre, stampidine exists as a mixture of two diastereoisomers. Molecular modelling studies and comparison of the lipase and protease-mediated hydrolysis rate constants indicated a chiral preference of the lipase active site for the putative S-stereoisomer.101104 Cellular metabolic studies of stampidine in COS-7 fibroblast cells revealed lipase or protease hydrolysis of the methyl ester group of the L-alanine side chain to form the cyclic intermediate in a stereoselective fashion.101,103 This intermediate was converted into the active metabolite, Ala-STV-MP. The superior antiviral activity of stampidine was attributed to the rapid formation of Ala-STV-MP. In addition, the presence of electron-withdrawing groups on the phenyl ring enhanced the activity of phosphoramidate derivatives by improving the rate of hydrolysis. Thus, the biological activity of these phosphoramidate derivatives is directly related to their rate of hydrolysis.

Solubility

Stampidine solubility in water was 3.0 mg/mL (Table 2). Solubility ranged from 4.6 mg/mL to 12 mg/mL in commonly utilized ophthalmic and topical formulations for human and veterinary use.108 In non-aqueous water miscible excipients containing polyethylene glycols and propylene glycol, the solubility was >20 mg/mL. The solubility values for stampidine with admixtures of varying amounts of water in hydroxylic excipients showed an exponential rather than a linear relationship.106,107 These solubility studies indicated that aqueous formulations of stampidine are feasible by adjusting the ratios of the excipients for preclinical and clinical studies. The presence of the nitrogen in the pyridine ring would be useful in preparing salt forms of stampidine for further improving its solubility for topical use.

Formulation

Development of stampidine as a topical agent for potential clinical use requires the evaluation of the design of appropriate ocular and genital drug delivery methods. Our initial work with stampidine has established its feasibility as a formulated drug product as a topical agent for mucosal bioavailability.105107 In a study of the accelerated excipient compatibility, stampidine was found to be stable in the presence of various formulation ingredients. The recovery of stampidine after 1 month of storage at 50°C in six excipients ranged from 71% to 94%.

Bioavailability

Stampidine was developed to overcome the dependence of STV on intracellular nucleoside kinase activation. Consequently, the dual-function antiviral activity of stampidine is substantially more potent that that of STV. The presence of a single para-bromine group in the phenyl moiety of stampidine contributes to its ability to undergo rapid hydrolysis yielding the key active metabolites Ala-STV-MP and STV with longer elimination half-lives.91,92,109 Micromolar plasma concentrations of the active metabolites of stampidine were rapidly achieved and maintained for more than 4 h after parenteral as well as oral administration of non-toxic doses of GMP-grade stampidine in preclinical studies.98,99


    Utility as an ocular antiviral agent
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
Stampidine is non-cytotoxic and non-irritating to mucosal epithelial cells.104106 Therefore, stampidine can be applied to the mucous membrane of the eye as an ointment or as an antiviral spray. In addition to purely fluid vehicles, such as aqueous solutions, suspensions and ointments, solid vehicles in the form of stampidine-releasing inserts can be utilized to deliver the antiviral agent to the eye surface to enhance the contact time. Current FDA standards for contact lens disinfecting systems require that viricidal activity be demonstrated against just one strain of herpes simplex virus (HSV), type 1.110 ADV may be more resistant to disinfection than HSV. Because of its greater water solubility, liquid aqueous ophthalmic compositions of stampidine will be suitable as a disinfectant for both hard and soft contact lenses contaminated with ADV. Stampidine will be useful as a surface coating of extended wear contact lenses, as a preventive method to prevent or inhibit ADV infections from occurring or biofilms from forming and as an ingredient of media used for the transportation and storage of human corneal transplants. As a genital microbicide, stampidine has been successfully formulated for topical use as a non-irritating vaginal microbicide.105108 Further evaluation of the anti-ADV activity of stampidine against adenoviral oculo-genital serotypes in vitro and in vivo along with the optimum concentration and dosing regimen may offer particular promise for the effective treatment of ocular as well as genital adenoviral infections.

The anti-adenoviral mechanism of action of stampidine is undefined but is probably not due to depletion of intracellular nucleotide pools that play an essential role in nucleic acid synthesis.111 Studies are currently in progress to assess the change in host cell RNA transcripts in response to virus-specific, stampidine-specific, and time-specific effects using DNA microarray (GeneChip Human Genome U95 set) that cross-examines the expression level of 12 625 genes. The effect of stampidine on the expression levels of genes associated with cell cycle regulation, apoptosis, oncogenesis, transcription, signalling and inflammation are being assessed using a Hierarchial Clustering algorithm. In agreement with our preclinical observations, stampidine did not affect the gene expression profile of inflammatory mediators. Our ongoing DNA microarray analysis has revealed that stampidine is a useful probe to develop new target sites relevant to the control of oculo-genital ADV/HIV pathogenesis.


    Conclusions
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
Adenoviruses are causative agents of contagious ocular and genital infections associated with conjunctivitis, genital ulcers and urethritis. Currently pursued anti-ADV drugs such as cidofovir and ribavirin are highly non-specific, suffer from toxic side effects, poor bioavailability and/or risk of emergence of drug-resistant strains. We discovered halogen-substituted phenyl phosphoramidate derivatives of STV as a new class of dual-function anti-HIV agents with potent and selective anti-ADV activity. Their antiviral potency is due to rapid formation of the key metabolite, Ala-STV-MP. The lead compound, stampidine, had remarkable in vitro and/or in vivo efficacy against drug-sensitive and drug-resistant HIV strains and ADV. As a result of its favourable animal toxicity profile, pharmacokinetics and in vivo antiretroviral activity, stampidine has clinical potential as an oculo-genital antiviral agent to inhibit, prevent or treat ADV/HIV infections.


    Acknowledgements
 
O. J. D. is supported by a research grant (2 R44 HD042884-02) from the National Institute of Child Health & Human Development, Bethesda, MD, USA. The work was conducted under NIH funding as well as Institutional Funds at the Parker Hughes Institute, which is a non-profit 501 (C) 3 tax exempt research organization with a public charity status. Institutional Animal Care and Use Committee's approval was in place for all the preclinical animal studies summarized in this review article.


    References
 Top
 Abstract
 Introduction
 Ocular and genital ADV...
 Ocular and genital ADV...
 Ocular manifestations of ADV...
 Limitations of current anti-ADV...
 Stampidine, a novel anti-ADV...
 Stampidine, a novel broad...
 Solubility and formulation...
 Utility as an ocular...
 Conclusions
 References
 
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