Abstract

A report published by the Centers for Disease Control concluded that some 65 million people in the US were infected with an sexually transmitted disease (STD) in the year 2000. While many of these diseases can be treated using antibiotics, viral infections, such as HSV, HPV and HIV, cannot. The resulting economic toll is staggering.

One potentially powerful method for the prevention of STDs is provided by topically applied microbicides. Unfortunately, compounds that exhibit high efficacy against infectious agents are often found to cause lesions in the vaginal epithelium, leaving women more vulnerable to infection. Monoglycerides (derived from breast milk) are non-toxic, and have been shown to posses powerful antimicrobial and antiviral activity which constitutes a defense system against infections that occur at mucosal surfaces.

The purpose of this study is to develop novel monoglycerides into microbicides for the prevention of STDs. The carbon chain length of these compounds will be optimized for activity. The various monoglyceride derivatives will be formulated into liposomal creams to enhance their bioavailability by providing a drug reservoir inside the vagina. Liposomes are especially suited for this task since they are composed of natural phospholipids and are, therefore, inherently safe. Safety and efficacy of these novel microbicides will be tested both in vitro and in vivo.

RESEARCH

A. Specific aims

The purpose of this study was to develop a novel microbicide for the prevention of sexually transmitted diseases (STDs). Milk lipids serve not only as nutrients but also as antimicrobial agents that constitute a defense system against microbial infections that occur at mucosal surfaces (1). The antimicrobial activity of milk lipids can be duplicated using purified fatty acids and monoglycerides. In vitro studies have demonstrated that one such monoglyceride, 2-O-octyl-sn-glycerol (OG), effectively kills gonorrhea and herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). It also inhibits infection of McCoy cells by human immunodeficiency virus (HIV). Therefore, these compounds are very promising for the prevention of STDs transmission.

Unfortunately, monoglycerides are poorly soluble in water. Moreover, aqueous formulations are poorly retained inside the vagina. Formulating these compounds into a vaginal cream, enhance their bioavailability by providing a reservoir that retains the drug inside the vagina over a period of 4-6 hours. Ideally, such a cream disperses rapidly during intercourse, thus allowing a high dose of drug to interact with infectious agents at the appropriate time. Liposomes are especially suited for this task. First of all they are composed of natural phospholipids and are, therefore, inherently safe. Liposomes are able to encapsulate lipophilic compounds in their membranes and protect them from the aqueous environment. Finally, they can be formulated into a cream, which will be easily suspended upon agitation.

Preliminary studies have suggested that monoacylglyceride derivatives with acyl chains longer than C8 may be more effective than OG (2). However, these active agents were never pursued due to their insolubility. Liposome based formulations provide a means to disperse and retain these agents in an aqueous environment. These studies were designed to identify the monoglyceride with the highest antimicrobial activity, and to optimize a liposomal formulation for this compound for use as a novel microbicide.

The supporting specific aims included:

Aim 1. Optimize the acyl chain length of the monoglyceride with respect to:

• Encapsulation efficiency of acyl-glycerol derivatives into liposomes
• Physical and chemical stability of the liposomal formulation
• Antimicrobial and antiviral potency of the formulation

Aim 2. Optimize liposome composition

• Determine the mechanism of action behind the killing of infectious agents
• Optimize antimicrobial effect by altering the liposome composition

Aim 3. Optimize the liposome-encapsulated monoglyceride formulation in vivo

• Evaluate safety in both ex vivo and in vivo models
• Evaluate the level of protection upon infectious agent challenge
• Optimize the viscosity of the formulation with respect to liposome retention and duration of protection

B. Background and Significance

1. Clinical need: Sexually transmitted diseases (STDs)

1.1 Prevalence of STDs

A report published by the Centers for Disease Control (CDC) in the year 2000 concluded that some 65 million people in the US are presently infected with an STD(3). The most common STD infections include chlamydia, gonorrhea, syphilis, herpes simplex type 2 (HSV-2), human papillomavirus (HPV), hepatitis B, and trichomoniasis. In addition to those already infected, it is estimated that every year in the US approximately 1 million people will contract HSV-2, 5.5 million will contract HPV, 3 million will contract chlamydia, and 40,000 will contract HIV(3).

While many of these diseases can be treated using antibiotics, viral infections, such as HSV and HIV, cannot. The resulting economic toll is staggering: In 1994, the CDC estimated that all STDs combined have an annual cost of approximately $17 billion (3). These costs include both indirect expenses (associated with lost productivity, job loss) and direct expenses (associated with cost of drugs for treatment, hospitalization, surgery, doctor's fees, etc.). Overall, HIV is by far the most costly of these diseases with an annual price tag of approximately $6.7 billion (3).

While women appear to be infected with STDs (excluding HIV) at about the same rate as men, they often face a greater health risk stemming from such infections. In many women, STDs are asymptomatic and may not be identified during routine examinations unless a specific test is performed. Without treatment, diseases such as gonorrhea and chlamydia may lead to the development of more serious medical problems including pelvic inflammatory disease, infertility, or ectopic pregnancy. More troubling are HPV (type 16) and HSV-2, both of which have no cure, and both of which have been linked to the development of cervical cancer. Many STDs also lead to complications with pregnancy and delivery. Approximately 30 to 40 % of all pre-term births and infant deaths appear to be linked to an STD infection(4).

Although HIV has been found to infect a disproportionately larger number of men compared to women in the past (due to the high rate of transmission through homosexual intercourse among gay men), rates of HIV infection in women is slowly becoming equal to that seen in men. In 1999, the number of females diagnosed with HIV made up 25% of the total population of individuals infected with HIV, up from the 1985 statistic of 7%. In the US, AIDS is now the fifth leading cause of death for all women between the ages of 25-44, and the third leading cause of death for African American women aged 25-44 (4).

1.2 Treatment and prevention of STDs

Many of the most common STD infections such as gonorrhea, syphilis, chancroid, chlamydia, and trichomoniasis may be treated with a single or multi-dose regimen of antibiotics or antimicrobials (5). Although these drugs are presently applied with a high rate of success, there has been an alarming trend in the resistance of several of organisms to some antibiotics. A good case in point is the increasing incidence of penicillin and tetracycline-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea (6). Consequently, there has been a switch to third generation cephalosporins and fluoroquinolones (i.e. azithromycin) in an effort to more effectively combat such infections. In all likelihood, these infectious agents will continue to be kept under control through the stringent use of antibiotics and antimicrobials.

In contrast to bacterial infections, there are no eradicative therapies presently available for viral STDs such as HSV-2, HIV, and HPV. Therapeutic agents have been developed to slow down the course of viral infection or to control viral outbreaks. These include viral DNA polymerase inhibitors as acyclovir, valaciclovir, and famicilovir for the control of genital herpes, and zidovudine, for the inhibition of HIV replication (7). Surgery and topical application of fluorouracil is used to control HPV, although neither treatment is capable of preventing future outbreaks. Additionally, it must be emphasized that while drugs and other methods of treatment are effective at slowing the spread of viral causative agents, they do nothing to prevent transmission of the disease to sexual partners.

The most effective means of combating the spread of viral STDs, and STDs in general is through prevention of the transmission to uninfected partners. Presently, the only effective means of prevention (barring abstinence) is the use of a physical barrier, such as a condom. However, in some situations it is difficult to negotiate the use of a condom and even when one is used, there still exists the possibility that infection will occur following breaking, leaking or loss of this protective barrier. Clearly, more options need to be identified to combat the spread of HIV and other STDs.

One potentially powerful method for the prevention of sexual transmission of pathogens is provided by topically applied microbicidal formulations (8). Microbicides for intravaginal use can prevent infection by interrupting one of three possible routes of pathogen transmission, and are grouped accordingly into three classes (9). The first group of compounds interacts with and disrupts the plasma membrane/viral envelop of the infectious agent thereby disabling or destroying the pathogen. These compounds include surfactants, peptides, plant extracts, and acid buffers. The second group includes compounds that directly inhibit viral entry by binding to receptors on the surface of host cells. These include the naphthalene sulphonate polymer PRO 2000, and sulphonated polysaccharides (including carrageenan and cellulose sulfate). The third class includes compounds that inhibit reverse transcription of the viral genome within the host cell. One compound belonging to this class is the reverse transcriptase inhibitor Tenovir.

1.3 Hurdles to the development of topical microbicides

Although the clinical need for safe and efficient microbicides is enormous (the number of females predicted to use such a product in the US alone is estimated at 21 million/year), a formulation is still 5 to 10 years from being introduced to the market (10, 11). The simplest explanation for this is that compounds that exhibit high efficacy against HIV and/or other causative agents are often found to also possess high toxicity. Toxicity to vaginal epithelium causes the formation of lesions, which leave women more vulnerable to infection (12). This has been most commonly seen with the detergent nonoxynol-9, which was originally developed as a spermicide but lately has undergone studies as a potential microbicide. A group of women who used nonoxynol-9 were actually found to have a greater number of new HIV infections than a control group (13).

Candidates from the other two classes of microbicides also have potential problems. These include viral entry through an alternative receptor or route (i.e. endocytosis), thereby circumventing receptors to which a drug has been developed. The third class of drugs, non-nucleoside reverse transcriptase inhibitors run the risk of becoming ineffective due to mutation of the viral enzyme.

Bearing in mind the potential problems associated with microbicides, it is important that new compounds continue to be developed and evaluated (14). Safety and efficacy criteria that must be met include low or no toxicity to vaginal epithelia and vaginal flora, non-teratogenic, high potency over a broad pH range, active and vehicle compatibility with latex, limited systemic absorption, and adequate intravaginal dispersion and retention (9, 15). One group of compounds that appears to satisfy these requirements includes monoglycerides. Several members of this group have been found to be non-toxic, while possessing potent anti-microbial and anti-viral activity.

2. Monoacylglycerides: A novel approach for prevention of STDs

2.1 Background

Over thirty years ago, it was observed that infants who were fed mother's breast milk were much less likely to suffer from gastrointestinal infections (16, 17). Further studies found that breast milk contained an antimicrobial factor, which was associated with the lipid fraction of milk, and which could be activated by enzymes found in the skim fraction (18, 19). These factors were later characterized as naturally occurring, medium chain saturated and long chain unsaturated fatty acids and monoglycerides. It was determined that these compounds had been produced by the degradation of milk triglycerides by lipoprotein lipase.

Subsequently, it was found that medium chain saturated and long chain unsaturated fatty acids and monoglycerides of these fatty acids were not only capable of killing several strains of gram positive and gram negative bacteria, but they were also effective at disrupting the envelope of visna virus, vesicular stomatitis virus (VSV), and herpes simplex 1 virus (HSV-1) (20-22). In 1994, Isaacs et al. found that medium chain lipids added to blood containing HIV-1 and HIV-2 were able to reduce the HIV titer by 1011 TCID50/mL (20). Such findings point to the possibility that naturally occurring fatty acids and monoglycerides can be used as intravaginal microbicides for the destruction of sexually transmitted infectious agents (1).

2.2 Chemistry

Since monoglycerides, such as monocaprylin, were found to have the greatest antiviral effect, they were subsequently used to develop a group of synthetic lipids for potential use as intravaginal microbicides (23). The synthetic lipids were designed to enhance water solubility and stability over that of naturally occurring fatty acids and monoglycerides, while retaining the antimicrobial properties of these chemicals. These synthetic lipids (structures shown below) included 1-O-hexyl-sn-glycerol, 2-O-hexyl-sn-glycerol, 1-O-heptyl-sn-glycerol, 2-O-heptyl-sn-glycerol, 1-O-octyl-sn-glycerol, and 2-O-octyl-sn-glycerol.

While all of these lipids have successfully been used to eliminate Escherichia coli, Salmonella enteritidis, Staphylococcus epidermidis, and Chlamydia trachomatis, 1- and 2-O-octyl-sn-glycerol were found to be the most effective at killing C. trachomatis (the causative agent in chlamydia infections). Specifically, 7.5 mM 2-O-octyl-sn-glycerol killed 100% of the Type D chlamydia serovar in 90 minutes of exposure, while 15 mM 1-O-octyl-sn-glycerol killed 100% of the bacteria in 30 minutes of exposure (23).

2.3. Application

The effort to develop more active derivatives of milk monoglycerides has lead to one major hurdle: the most active compounds are poorly water soluble. A solution to this problem is to provide a drug delivery vehicle.

Liposomes have a number of properties which make them suitable for use as drug carriers: They are composed of natural constituents (phospholipids) and are, therefore, biodegradable and have very little inherent toxicity. The lipid phase of the bilayer provides a non-polar environment that is ideal for sequestering hydrophobic drugs, such as octylglycerol (OG) and other monoglycerides. Liposomes can be easily dispersed in aqueous environments, making them an effective tool for "solubilizing" and distributing poorly water-soluble drugs. In addition, liposomes can be formulated into a cream, which provides a reservoir of drug inside the vagina.

C. Preliminary Data

1. Liposome mediated monoglyceride delivery

Development of a liposome-encapsulated formulation of octylglycerol, was undertaken in an effort to provide proof of concept that this drug can indeed be encapsulated into liposomes yielding stable formulations that are biologically active and non-toxic.

1.1 Experiment I: Effect of lipid concentration

Liposome formulations containing 0.5 wt % octylglycerol were prepared using 1, 2.5, 5, and 10 wt % phosphatidylcholine (PC). The anti-microbial activity of the formulations was tested on several strains of gonorrhea, HSV-1, HSV-2, and HIV. In addition, the formulations were tested for safety towards naturally occurring vaginal flora, such as Lactobacillus. Formulations without OG served as negative controls. All octylglycerol containing formulations were found to effectively kill all strains of gonococcus tested as well as HSV-1 and HSV-2 (Table I). Infection of McCoy cells by HIV appeared to be suppressed by all liposome-encapsulated octylglycerol formulations (Table I), but not by the controls. It was found that 1% PC + 0.5% OG suppresses 90% of the HIV infection as compared to 10% PC + 0.5% OG, which appeared to suppress only 80% of HIV infection. This suggests an inverse correlation between the amount of lipid and the activity of the formulation. This can be explained by the fact that less drug is present per liposome. None of the negative control formulations without OG appeared to kill gonococcus or HSV, nor was it found to suppress HIV infection in McCoy monolayers. This shows the activity was due to the OG and not due to the delivery vehicle.

None of the formulations were found to be toxic to Lactobacillus.

Table I. Antimicrobial activity of liposome-encapsulated 0.5 wt % OG in relation to PC content. The “+” indicates a reduction in infectivity of at least 1,000 fold. In case of HIV, the % inhibition of infection is indicated. None of the controls formulations without OG displayed any killing or inhibition of infection.

Conclusions

• Formulations containing 0.5% OG effectively kill gonorrhea, and suppress infection by HSV-1 and -2, and HIV.
• The phospholipid concentration has no apparent effect on the ability of OG-containing formulations to kill gonorrhea, HSV-1 or HSV-2, however, there appears to be an inverse correlation of HIV inhibition and lipid concentration. For example, the formulation containing 10% PC suppresses 80% of HIV infection, while the 1% PC formulation suppresses HIV infection by 90%.
• Octylglycerol-encapsulated liposomes do not appear to be toxic to Lactobacillus.

1.2 Experiment II: Octylglycerol dose response study

The concentration of octylglycerol which was most effective at killing infectious organisms was determined using formulations containing 10% PC and 0.1, 0.3, 1, 3, and 6% octylglycerol, in citrate buffer pH 5. In addition, the formulations contained the preservatives methylparaben (0.45 wt%) and propylparaben (0.1 wt%). These preservatives had previously been shown to be non-toxic to Lactobacillus. The 10% PC formulation was chosen because it was a pharmaceutically elegant formulation. It had a viscosity similar to that found in commercially available products for vaginal use (i.e. lubricants, spermicides). These formulations were tested on the five gonococci strains, HSV-1, HSV-2, and HIV. In addition the safety of these formulations towards Lactobacillus was tested. The results are presented in Table II.

Table II. Dose response of liposome-encapsulated OG. Liposome formulations containing 10% PC and various amounts of OG were tested for antimicrobial activity. The “+” indicates a reduction in infectivity of at least 1,000 fold. In case of HIV, the % inhibition of infection is indicated. None of the controls without OG displayed any killing or inhibition of infection.

Conclusions

• Formulations containing 1 - 6% OG appeared to be effective at killing all strains of gonorrhea tested, HSV- 1 and –2, and HIV.
• The killing of HIV appeared to increase with increasing OG concentration.
• Two highest OG concentrations (3 and 6%) appeared to coincide with an increased toxicity to the mammalian cells used for the HIV test.

D. Research Design and Methods

Aim 1: Optimize the acyl chain length of the monoglyceride

1.1 Rationale

It has been shown that there is a direct correlation between the length of the acyl chain of naturally occurring fatty acids and monoglycerides and their antiviral and antibacterial activity (2, 23). Fatty acids and monoglycerides with either short or long acyl chains are not as effective at killing microbes as medium chain monoglycerides, such as monocapryloyl (C8), monocaprin (C10), and monolaurin (C12) (2). This parabolic structure-function relationship is not unusual, and has been observed with many other putative antimicrobial agents including a number of acylcarnitine analogues and quaternary ammonium L-carnitine esters (24, 25).

Several synthetic monoacylglycerides were designed based on the structure of monocapryloyl (C8), and tested for their effectiveness at killing Chlamydia trachotamatis. These derivatives included C6, C7, and C8 monoglycerides. Of these, the C8 derivative was found to kill C. trachotamatis most effectively, while the C7 and C6 derivatives were correspondingly less potent (23). Although results obtained with naturally occurring monoglycerides suggested that monoacyl derivatives with chain lengths greater than C8 and less than C12 would be the most potent of all, such derivatives have never been synthesized or tested.

The studies described investigate the antimicrobial activity of synthetic medium chain monoglyceride derivatives (C8, C10, C12 and C14). The formulation problems that occur with these hydrophobic compounds will be solved by encapsulation into liposomes. Preliminary results have already shown that liposomes can encapsulated octylglycerol efficiently, without loss of activity. The liposome-encapsulated monoglyceride formulations will be characterized with respect to their the physical and chemical stability, and their anti-microbial effectiveness in vitro

Optimization of liposome composition

2.1 Rationale

Understanding the mechanisms behind the interaction of liposomes and infectious agents is important for the optimization of the formulation. Two possible mechanisms exist. The first mechanism proposes that the monoglyceride must be released from the liposome, i.e. free in solution, in order to be effective. The second mechanism proposes that the monoglyceride is delivered directly to the infectious agent through fusion of the liposomal membrane with the bacterial membrane or viral envelope. Once the mechanism of action is elucidated, liposomes may be optimized to increase their activity.

2.2 Experimental plan

_____2.2.1 Defining the mechanism of action

The amount of free drug in solution and the release rate of monoglyceride from the liposomes into solution will be determined by dialysis. The concentration of free drug in the dialysate will be determined using the GC method described in section 1.2.1. Monoglycerides free in solution will serve as a positive control.

To study if the liposomes interact directly with the infectious agent, liposomal membranes will be labeled with the fluorophore 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (pyrPC)at a mol ratio of 0.85:0.15 (PC:pyrPC). Upon membrane fusion between labeled liposomes and the unlabelled viral particles, there is a substantial drop in the fluorescent signal, which can be measured using a fluorimeter (28, 29). The signal will be corrected for fluorescence decrease caused by dilution.

_____2.2.2 Optimize antimicrobial effect of monoglyceride containing liposomes

Once the mechanism of action is elucidated, the liposomes may be optimized to increase their activity. If it has been shown that the drug must first dissociate from the liposome prior to pathogen interaction, increased monoglyceride release may be achieved by increasing the membrane fluidity. The effect of adding a fatty acids (e.g. oleic acid) on formulation activity will be evaluated. Alternatively, if it is found that membrane fusion must occur to elicit an antimicrobial effect, the addition of cholesterol or sphingolipids may enhance the fusogenic properties of the liposomal membranes (30-33). The antimicrobial activity of the modified liposomes will be assessed as described in section C.1.1.

2.3 Anticipated results and contingencies

The first sub-aim in this section establishes the mechanism by which monoglyceride interacts with infectious agents. Preliminary results have already shown very little free octylglycerol suggesting that membrane fusion may play an important role. Regardless of the mechanism of action, these experiments are essential to rationalize the optimization process for the liposome mediated antimicrobial effect.

E. References