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Ultra-long-acting removable drug delivery system for HIV treatment and prevention/Dolutegravir Implant
 
 
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"To address this significant limitation and to extend the duration of treatment, an ultra-LA injectable and removable formulation for HIV PrEP based on in situ forming implant technology was developed."
 
One step closer to ultra-long-acting PREP?
 
Harper, Kirsten Nicole
 
AIDS: march 2019
 
Pre-exposure prophylaxis (PrEP) has become an increasingly popular means of preventing HIV transmission, but the only currently approved form requires users to take a daily pill. Long-acting forms of PrEP could offer a simpler regimen, potentially improving adherence and effectiveness. One potential concern, however, is how clinicians could respond to side effects that emerge after a long-acting form of PrEP is delivered; at present, formulations in late-stage development cannot be removed from the body once they are injected. Recently, researchers published findings from a preclinical study of a novel type of ultra-long-acting, removable PrEP in Nature Communications [1].
 
If validated in future studies, 'this form of PrEP could provide a viable alternative for the numerous individuals who would prefer long-term protection without the need for daily dosing,' says Martina Kovarova, PhD, of the University of North Carolina at Chapel Hill, lead author of the article. The implant is delivered via subcutaneous injection of a solution containing the antiviral dolutegravir; after being injected, the solution quickly solidifies into a small biodegradable implant that can be removed if problems arise. In the study, researchers showed that a single implant delivered sustained levels of dolutegravir for up to 9 months in two mouse models [NSG mice and humanized bone marrow/liver/thymus (BLT) mice] and for up to 5 months in macaques. In addition, plasma collected from mice and macaques with the implant was able to inhibit HIV-1 replication ex vivo, and HIV replication was inhibited in BLT mice with the implant in vivo. Finally, the implant was able to protect BLT mice from multiple high-dose vaginal challenges with HIV. Raphael J. Landovitz, MD, MSc, of UCLA Health in Los Angeles, says that although this technology is exciting, it is still too early to know how much promise it holds for humans. First, the safety of dolutegravir for women of childbearing potential is currently being evaluated. Second, it will be important to investigate how pharmacokinetics vary in response to factors such as sex at birth and BMI. Finally, he notes that during the component of the study simulating HIV treatment, viral resistance to dolutegravir monotherapy developed quickly. This suggests that, if intended for treatment, a future version of the implant would need to include additional antiviral agents, though this might not be necessary for PrEP.
 
While agreeing that there is much work left to do before the implant is ready for testing in humans, J. Victor Garcia, PhD, senior author of the article and also from the University of North Carolina at Chapel Hill, is optimistic. Referring to the biodegradable implant that forms after injection, he says, 'The fact that similar technology has already been used in the clinic for other types of applications, such as palliative treatment for advanced prostate cancer, increases our optimism that this form of PrEP can be used for HIV prevention.'
 
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Ultra-long-acting removable drug delivery system for HIV treatment and prevention
 
Nature Comm.. Oct 2018- Martina Kovarova1, S. Rahima Benhabbour2, Ivana Massud3, Rae Ann Spagnuolo1, Brianna Skinner4, Caroline E. Baker1, Craig Sykes5, Katie R. Mollan6, Angela D. M. Kashuba5, J. Gerardo Garcia-Lerma3, Russell J. Mumper2 & J. Victor Garcia1
 
Abstract
 
Non-adherence to medication is an important health care problem, especially in the treatment of chronic conditions. Injectable long-acting (LA) formulations of antiretrovirals (ARVs) represent a viable alternative to improve adherence to HIV/AIDS treatment and prevention. However, the LA-ARV formulations currently in clinical trials cannot be removed after administration even if adverse events occur. Here we show an ultra-LA removable system that delivers drug for up to 9 months and can be safely removed to stop drug delivery. We use two pre-clinical models for HIV transmission and treatment, non-human primates (NHP) and humanized BLT (bone marrow/liver/thymus) mice and show a single dose of subcutaneously administered ultra-LA dolutegravir effectively delivers the drug in both models and show suppression of viremia and protection from multiple high-dose vaginal HIV challenges in BLT mice. This approach represents a potentially effective strategy for the ultra-LA drug delivery with multiple possible therapeutic applications.
 
Introduction
 
Adherence to medication is essential to treatment success1. In most cases the extent to which patients are able to follow prescribed treatments determines the final outcome. This is particularly important in the treatment of chronic conditions like mental illnesses, hypertension, diabetes, and HIV/AIDS1. Sustained drug release has successfully improved adherence in patients with schizophrenia2 and as contraceptives3. Long-acting (LA) injectable formulations of ARV can increase adherence and effectiveness of HIV treatment and prevention4. In particular, LA formulations can (1) simplify dosing schedules, (2) reduce possible side effects, (3) provide constant concentration of drug, and (4) have a positive effect on patient's overall quality of life. LA-ARV formulations currently in clinical trials, are formulated as nanosuspensions for injections every 8 weeks5,6,7 and it is impossible to remove the injected nanosuspension from the body in the case of a medical emergency.
 
To address this significant limitation and to extend the duration of treatment, an ultra-LA injectable and removable formulation for HIV PrEP based on in situ forming implant technology was developed. This allows drug administration by subcutaneous injection followed by implant solidification in vivo and subsequent biodegradation of the implant resulting in sustained drug release8,9. The efficacy of drug delivery via ultra-LA formulation in the context of HIV is evaluated in BLT humanized mice, systemically reconstituted with human hematopoietic cells10. BLT mice have been extensively used for HIV transmission, replication, and persistence studies11,12,13,14. Importantly, humanized BLT mice allow evaluation of HIV treatment and prevention strategies with a variety of transmitted/founder HIV-1 isolates via relevant routes of transmission.
 
Results
 
Preparation of ultra-LA dolutegravir

 
Dolutegravir, a highly effective second-generation HIV integrase strand transfer inhibitor with extensive track record of efficacy and safety15, was used for preparation of ultra-LA formulation. In addition to active drug, the formulation consists of two relatively low cost FDA-approved excipients: (1) poly(lactic-co-glycolic acid) (PLGA), a biodegradable copolymer that eventually and safely biodegrades and, (2) N-methyl-2-pyrrolidone (NMP), a water miscible and biocompatible organic solvent16,17. Composition of the ultra-LA formulation was first optimized for release kinetics in vitro and then sustained delivery of dolutegravir in vivo (Fig. 1). The formulation contained dolutegravir/PLGA/NMP at a ratio 0.3:1:2 by weight, had a viscosity 845 cP at 25 °C (Brookfield Cone and Plate Digital Rheometer, n = 3). Formulation stability was assessed as the ability to remain in solution (measured by dynamic light scattering, Zetasizer Nano ZS Particle Analyzer), and to maintain a stable dolutegravir concentration (98% of the original concentration) measured by HPLC analysis. Dolutegravir was chemically stable in this formulation at 25 °C for at least 6 months. In the aqueous environment in vitro (0.01 M PBS, pH = 7.4, 2% solutol) solidification of the implant was instantaneous.
 
Pharmacokinetics of ultra-LA dolutegravir
 
For an initial in vivo evaluation, anesthetized female NSG and BLT mice received a single subcutaneous injection of the ultra-LA dolutegravir on their back (5.5-7.0 mg dolutegravir in 50-80 µl). The injected formulation first formed a hard translucent globule under the skin that turned yellowish white (within 48 h of administration) as the formulation solidified. The formulation was well tolerated by the mice and no injection site reactions or other signs of overt toxicity, changes in behavior, movement, water consumption or weight loss were noted. For the purpose of observing the nature of the implant and to confirm that it could be readily removed from the mice, 1 week after administration a small incision was made near the location of the implant in one of the mice allowing rapid removal of the implant from the mouse (Fig. 1a). The rest of the mice were used for in vivo pharmacokinetic analysis of drug release (Fig. 1b). Plasma concentrations of dolutegravir were quantitated using a validated high-performance liquid chromatography-tandem mass spectrometry LC/MS-MS method18. Non-compartmental analysis of the median composite pharmacokinetic (PK) profile demonstrated a biexponential decay. After an initial 1st order decline in plasma concentrations, the release of dolutegravir approached zero-order kinetics. Plasma concentration of dolutegravir was ten times greater than the protein adjusted (PA)-IC90 for at least 5 months post administration. Even at 283 days after ultra-LA dolutegravir administration 1/3 mice still had detectable dolutegravir in plasma. We then used a sparse PK analysis to compare the concentration of plasma dolutegravir between BLT humanized and non-humanized mice. Our results demonstrated similar pharmacokinetics of dolutegravir in both types of mice (Supplementary Fig. 1). Concentration of dolutegravir was also evaluated in the female reproductive tract (FRT) in 14 NSG mice receiving a single subcutaneous injection of ultra-LA dolutegravir. Vagina, cervix, uterus, and plasma from treated mice were collected 1, 4, and 12 weeks post administration and dolutegravir concentrations determined (Fig. 1c). One week after ultra-LA dolutegravir administration, the median concentration of dolutegravir in plasma, vagina, cervix, and uterus were 1350 ng/ml, 196 ng/mg, 158 ng/mg, and 272 ng/ml, respectively. One month post administration, the median dolutegravir concentrations were 958 ng/ml, 233 ng/mg, 262 ng/mg, and 303, ng/mg, respectively, and 12 weeks post administration the median concentrations were 1200 ng/ml, 356 ng/mg, 170 ng/mg, and 284 ng/mg, respectively (Fig. 1c, Table 1). Differences in DTG concentrations within each compartment (vagina, cervix, uterus, and plasma) comparing 1 week, 4 weeks, and 12 weeks (n = 6 per group) did not reach statistical significance (Kruskal-Wallis test plasma p = 0.21, cervix p = 0.09, uterus p = 0.70, vagina p = 0.17). Observations were combined over weeks 1, 4, and 12 to evaluate whether plasma concentrations were higher than tissue concentrations for each tissue type separately. Dolutegravir concentrations in plasma were higher than in tissue for each of the three tissue types for every animal (Wilcoxon signed-rank p < 0.001 for cervix, uterus, and vagina analyses, respectively). Together, these results demonstrate the sustained in vivo release of dolutegravir into plasma and its efficient penetration into tissues of the female reproductive tract.
 
To assess safety and drug release profile of the ultra-long-acting formulation of dolutegravir in a large animal model, two rhesus macaques were subcutaneously administered the ultra-LA dolutegravir formulation (100 mg). Animals were monitored for signs of toxicity and skin reactions at the injection site, including erythema, edema, and hematoma formation, presence of induration, and any other lesions such as abscesses, necrosis, dehiscence, or local inflammation twice a week. The implants were well tolerated with little or no sign of toxicity for 5 months (last point analyzed). Administration of the ultra-long-acting formulation of dolutegravir resulted in sustained dolutegravir concentration in plasma for more than 140 days (Fig. 1d). These results demonstrate the feasibility of ultra-long-acting dolutegravir delivery system for sustained delivery in rhesus macaques. However, this formulation originally developed for BLT mice will have to be further optimized prior to efficacy studies in macaques and its potential future applications in humans.
 
Inhibition of HIV-1 replication ex vivo
 
Having demonstrated sustained concentration of dolutegravir in plasma of both mice and macaques and in tissues from mice we proceeded to evaluate its antiviral activity. Serum obtained from the ultra-LA dolutegravir-treated mice in Fig. 1c demonstrated strong, concentration dependent antiviral activity (Fig. 2a). Specifically, a one hundredth-fold dilution of serum was able to block in vitro viral infection by >86% when collected 7 or 28 days post administration (median 89%, range 86.4%-90.8% and median 86%, range 74.1%-87.9% for 7 and 28 days, respectively). A 100-fold dilution of serum was also able to block HIV infection by 66% when collected 84 days post ultra-LA dolutegravir administration (range 63.5%-67.0%) (Fig. 2b). Statistical analysis of the antiviral activity present in serum demonstrated a strong correlation with dolutegravir concentrations (Kendall rank correlation coefficient 0.75; 95% CI: 0.65-0.85) (Fig. 2c).
 
Inhibition of HIV-1 replication in vivo
 
To establish the in vivo inhibitory effect of dolutegravir administered via the ultra-LA dolutegravir formulation on HIV replication, eight BLT humanized mice were first infected intravenously with HIV-1JR-CSF (3 x 104 TCIU) (Fig. 3a, Table 2). HIV-RNA was readily detected in plasma from all exposed mice 2 weeks after exposure (median 1.97 x 106 copies/ml, range 0.65-8.50 x 106copies/ml). Two weeks post infection, mice received a single dose of ultra-LA dolutegravir- or placebo administered via subcutaneous injection (n = 4 for each group). Dolutegravir plasma concentrations in treated mice were sustained throughout the entire experiment (Fig. 3b). Placebo-treated mice maintained high concentration of plasma HIV-RNA. In contrast, strong suppression of HIV replication (∼1-2 log) was noted in all mice treated with the ultra-LA dolutegravir formulation (Fig. 3c, d). In one mouse, viral load fell below the level of quantitation (LOQ, 1375 copies of HIV-RNA/ml) as early as 2 weeks post administration (Fig. 3c). Plasma viral load AUC was smaller in the ultra-long-acting dolutegravir group compared to the placebo group, p = 0.03 (exact Wilcoxon-Mann-Whitney test, n = 8). Virus RNA from plasma of mice treated with ultra-LA dolutegravir for 19 and 50 days were sequenced to determine whether mutations associated with drug resistance were acquired during this course of dolutegravir monotherapy. At day 19 bulk RNA sequences were obtained from the three mice with detectable viral loads. All three had a single nucleotide substitution resulting in an amino acid change at position 157 (E→K). At day 50, viral RNA from all four-treated mice was analyzed. At this time point, all three mice analyzed at day 19 had mutated from 157K to 157Q. In addition, in one mouse a mutation was also detected at position 263 (R→K). On day 50, the viral RNA from the mouse that was below detection at day 19 and thus could not be analyzed, had a mutation at position 157 (E→K). Sequencing of individual clones from all viral RNAs obtained at days 19 and 50 revealed the presence of several other mutations (Table 3). The majority of them were naturally occurring polymorphic substitutions. Interestingly, both the R263K and E157Q mutations were found in 2/8 clones from one mouse. Together, these two mutations have been shown to increase the resistance of HIV to dolutegravir in vitro19.
 
To determine the effect of ultra-LA dolutegravir on the concentration of HIV-RNA in the female reproductive tract mice were lavaged at the indicated time points and samples analyzed for the presence of HIV-RNA and dolutegravir concentrations. Within 2-3 weeks of treatment the concentration of HIV RNA in cervico-vaginal secretions (CVS) rapidly decreased below LOQ (81 HIV-RNA copies per 60 µl) with transient low-level viral increases in two mice (Fig. 3e, f). These results indicate that despite the observed lower concentration of dolutegravir in the FRT compared to plasma (Table 1), the dolutegravir concentration in FRT was sufficient to efficiently suppress viral replication (Table 2). A slow decrease in CVS HIV-RNA was also observed in the placebo-treated mice, most likely as a result of the dramatic reduction in CD4 T-cell numbers occurring during HIV infection in the FRT (Fig. 3h)11. In contrast to the dramatic depletion of CD4+ T cells noted in CVS from control (i.e., not treated) mice, in ultra-LA dolutegravir-treated mice, CD4 T-cell numbers progressively increased to the pre-exposure levels. CD4+ T-cell percentage AUC was larger for all mice in the ultra-long-acting dolutegravir group compared to the control group, p = 0.03 (exact Wilcoxon-Mann-Whitney test, n = 8) further demonstrating the efficacy of ultra-LA dolutegravir treatment on systemic HIV infection (Fig. 3g, h).
 
Protection from vaginal HIV acquisition
 
To evaluate the potential of the ultra-LA dolutegravir to prevent vaginal HIV transmission, BLT mice (n = 5 per group) were subcutaneously administered ultra-LA dolutegravir (treated mice) or placebo (control mice) (Fig. 4a). Seven days later, mice were challenged vaginally with a high dose of one of two transmitted/founder viruses HIV-1CH040 (3.0 x 105 TCIU, 2 controls, three treated mice) or HIV-1THRO (3.5 x 105, three controls, two treated mice). The two control mice exposed to HIV-1CH040 became plasma HIV-RNA positive within 2 weeks after challenge and 2/3 control mice challenged with HIV-1THRO became plasma HIV-RNA positive within 3 weeks after the challenge (Fig. 4b). All ultra-LA dolutegravir-treated mice remained HIV negative after the first exposure. Six weeks after first challenge, ultra-LA dolutegravir-treated mice were challenged vaginally a second time with a high dose of HIV-1CH040 (3.0 x 105 TCIU). One-treated mouse became HIV-RNA positive 1 week after the second challenge, and sequence analysis identified the breakthrough virus as HIV-1CH040 with no mutations in the HIV integrase gene. All other treated mice (4/5) remained negative for plasma HIV-RNA (Fig. 4d, Table 4). Two different but complementary approaches were used to exclude the possibility that HIV-1 was transmitted but suppressed due to the continuous presence of dolutegravir. First, two mice without evidence of HIV-RNA in plasma were collected 11 and 13 weeks post ultra-LA dolutegravir administration for tissue HIV-DNA analysis. No evidence of HIV DNA was noted in any of the tissues analyzed confirming systemic protection from infection. Second, the ultra-LA dolutegravir implant was surgically removed in the remaining two HIV-1-negative mice 15 weeks post-implantation. Implant removal resulted in a rapid decrease of plasma dolutegravir concentrations (>1 log within first 3 days after the removal) (Fig. 4f). After implant removal, no HIV-RNA was detected in plasma (Fig. 4c). Four weeks post-removal of the ultra-LA dolutegravir mice were killed, tissues were collected and analyzed for the presence of cell-associated HIV-DNA. No evidence of HIV-DNA was found in any of the tissues analyzed confirming that the lack of viremia after ultra-LA dolutegravir removal was not due to an occult infection masked by the continuous presence of high concentration of drug (Table 4). Rather these results strongly indicate that these four mice were fully protected from two high dose vaginal exposures to HIV 6 weeks apart. In order to establish a possible cause for the single breakthrough infection noted above, plasma drug concentrations throughout the course of the experiment were analyzed. The concentration of dolutegravir in plasma of 4/5 treated mice was maintained at 2805 ng/ml (median, range 1510-4510 ng/ml) for 11 weeks. In the mouse with the breakthrough infection, plasma dolutegravir concentration began to slowly decrease 3 weeks after injection. At the time of the second viral challenge, the plasma dolutegravir concentration was 659 ng/ml. Plasma dolutegravir concentration continued to drop and by 11 weeks post ultra-LA dolutegravir administration it was below 10 ng/ml (Fig. 4e). These results suggest that the breakthrough infection is likely due to the lower concentrations of dolutegravir in this mouse.
 
Discussion
 
The HIV epidemic continues to be a significant health concern worldwide. In 2016, ∼36.7 million people were living with HIV20. Among promising preventive interventions is pre-exposure prophylaxis (PrEP), in which ARVs are taken by HIV-negative people before potential exposure to the virus. Clinical studies established that daily oral PrEP with Truvada® can prevent HIV infection among high-risk populations21,22,23,24,25. However, recent clinical trials demonstrate that lack of adherence to the indicated drug regimen is common, resulting in lack of protection from HIV infection26,27.
 
In the specific case of HIV prevention, the lack of adherence by clinical trial participants has served to highlight the urgent need for drug delivery systems capable of offering long-term protection from HIV infection26,27. Two LA-ARV formulations for HIV PrEP are in clinical trials. Cabotegravir, an integrase strand transfer inhibitor (INSTI), is in phase IIa and IIb/III clinical trials28 and rilpivirine, a non-nucleoside reverse transcriptase inhibitor (NNRTI), is in a phase II clinical trial29. Both drugs are formulated as nanosuspensions for LA intramuscular injections every 8 weeks5,6,7. Major limitations of this type of drug formulations are the complete inability to remove the injected nanosuspension from the body in the case of a medical emergency and the inability of co-formulation of two or more drugs. Removal of LA formulations is essential to circumvent adverse reactions or to prevent long-term sub-therapeutic drug exposure after PrEP discontinuation. These concerns are currently being partially addressed by an oral regimen of drug for 5 weeks prior to the administration of the LA formulation and by daily oral administration of Truvada for up to 1 year at the end of a dosing regimen7. Given the documented evidence of lack of compliance with this approach, these options are likely not appropriate for the targeted populations.
 
Humanized mouse models were used for pre-clinical efficacy assessment of LA formulations of ARVs. A nanosuspension of crystalline rilpivirine, administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge 1 week after drug administration and provide partial protection 4 weeks after drug administration12. Similarly, single subcutaneous dose of LA raltegravir significantly protected BLT mice from vaginal HIV acquisition 4 weeks after drug administration13. Recently, a lipophilic modified DTG prodrug encapsulated into poloxamer nanoformulations significantly protected CD34+ humanized mice from the HIV-1 for 2 weeks30. These results suggest utility of humanized mouse models for pre-clinical evaluation of LA formulation of AVR.
 
Optimally, a LA delivery system for HIV prophylaxis should be effective, safe, easy to apply, and affordable so that it can be used in resource-poor and constrained settings. The data in this paper indicate that ultra-LA dolutegravir can be readily prepared and sterilized by filtration. It can efficiently deliver dolutegravir for up to 9 months in mice and 140 days in NHP (last time point analyzed). A single administration of ultra-LA dolutegravir strongly inhibited acute HIV replication, and effectively protected against repeated high-dose vaginal HIV challenges using highly relevant primary transmitted/founder viruses. An important aspect of the ultra-LA dolutegravir formulation is the fact that it can be easily removed resulting in rapid decrease of drug concentration providing a measure of safety that is not afforded by any of the current LA drug delivery systems in clinical trials for HIV prevention. However, when immediate removal is not necessary, the formulation is biodegradable and does not require surgical removal. In addition, the formulations offer the flexibility to include multiple drugs and the future potential to be reloadable in situ31.
 
In this stage of formulation development, it is difficult to predict how much ultra-LA dolutegravir will be needed to achieve same efficacy of prevention from HIV transmission as seen in BLT mice. However, we can compare this formulation to existing LA formulations in clinical trials. Cabotegravir nanoformulation consists of two 2-ml injections of product containing 200 mg/ml drug administered every 2 months. Our goal is to develop a formulation that can accommodate 250 mg/ml dolutegravir. As the IC90 for dolutegravir (64 ng/ml) is 2.5-fold lower that the IC90 for cabotegravir (166 ng/ml) we consider this to be an appropriate target. PK analysis shown in Fig. 1 also suggests longer sustained release of dolutegravir in vivo compared to the current cabotegravir formulation. Therefore, using the same parameters currently used for cabotegravir, our ultra-LA dolutegravir is likely scalable to humans.
 
This technology was inspired from the Atrigel product that is commercially available as Eligard (please see citation 8 and 9). This is a palliative treatment for advanced prostate cancer. However, there are several differences between Eligard and our ultra-LA delivery system. (1) Eligard is packaged as a 2-syringe system due to the limited stability of leuprolide acetate in the PLGA/NMP formulation and administered as a dispersion rather than a solution32. (2) The ultra-LA dolutegravir formulation presented in this manuscript uses an antiretroviral drug instead of a synthetic hormone, (3) the formulation is prepared as a solution that is stable for more than 6 months at room temperature. Since the Atrigel technology has already been approved by the FDA for treatment of prostate cancer we anticipate that it is also applicable for HIV PrEP. Several key observations are worth highlighting. Dolutegravir monotherapy resulted in strong and sustained suppression of HIV replication. However, as early as 19 days post therapy initiation, resistance mutations begun to appear in the replicating viruses. By day 50 post therapy initiation, both drug resistance and compensatory mutations were found. Consistent with studies in women18, the concentration of dolutegravir in FRT tissues from BLT mice was 3-7 times lower than in plasma. However, even under these conditions significant protection from high dose vaginal infection was noted. The one case of breakthrough infection could be attributed to the lower drug concentration present in this mouse at or near the time of exposure (659 ng/ml). Of note, the dolutegravir plasma concentrations sustained in the mice protected from high dose HIV infection (∼2800 ng/ml) are directly comparable to those of cabotegravir reported to be protective in a non-human primate model of low dose repeated exposure vaginal SHIV infection33. In summary, the results demonstrate the in vivo effectiveness of an ultra-LA formulation of dolutegravir to deliver drug for up to 9 months that results in sustained viral suppression and prolonged protection from high dose HIV vaginal challenges. With its ultra-long duration, low cost of production, ease of administration and the ability to be removed this represents a significant advance in drug delivery for HIV pre-exposure prophylaxis.

 
 
 
 
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