Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis

Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18–24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.


Results
Intrinsic anti-Wolbachia potency of rifampicin is superior to doxycycline. Table 1 shows in its first row, the in-vitro IC 50 levels obtained for doxycycline and rifampicin in the Wolbachia infected C6/36 cell (C6/36 wAlbB) assay 57 . Rifampicin exhibited an EC 50 of 1.3 nM, which was approximately 16.2 fold more potent than that of doxycycline (EC 50 = 22 nM), against Wolbachia. Both drugs resulted in killing ~90% of Wolbachia in comparison to vehicle control cell cultures at the end of the 7 day experiment.
Pharmacokinetics of rifampicin are superior to doxycycline. Figure 1 shows the pharmacokinetic profiles measured in SCID mice at days 1 and 7 for doxycycline or rifampicin at a dose of 25 mg/kg bid. Table 2 summarises the pharmacokinetic parameters of each drug. Rifampicin (CL/F = 0.11 L/hr/kg) exhibited ~10 fold higher exposure at the same dose than doxycycline (CL/F = 1.2 L/h/kg). Clearance values at day 1 and day 7 were not statistically different (Mann-Whitney test, n = 5 mice, P > 0.05) and as shown in Table 2 all pharmacokinetics parameters of both drugs did not significantly change after chronic exposure in comparison to single dose administration.

Drug
Doxycycline Rifampicin

Ratio (doxycycline/ rifampicin)
in-vitro IC 50 (mg/mL) 22 1.3 16.9 in-vivo IC 50 (µg/mL) 1059 65 16.3 Table 1. In-vitro and in-vivo IC50 values of doxycycline and rifampicin calculated based on the individual potency of each drug in the dose escalation studies in animals using PK-PD modelling.
Scientific REPORTS | _#####################_ | DOI: 10.1038/s41598-017-00322-5 Anti-Wolbachia activity of rifampicin is superior to doxycycline in adult filarial infection models. We compared the anti-Wolbachia activity of rifampicin (5 mg/kg qd, 15 mg/kg qd, 35 mg/kg qd or 25 mg/ kg bid) with doxycycline (25 mg/kg bid) in B. malayi and O. ochengi SCID mouse models (Fig. 2). The murine doxycycline dose had been previously defined as bioequivalent to clinical dosing with 100 mg/day 58 . Wolbachia rate-of-kill was examined by four day dosing of mice infected with adult-stage B. malayi followed by termination +24 h after last dose and retrieval of female adults for assessment of Wolbachia loads (Fig. 2a). Changes in Wolbachia post-dosing were calculated as a % reduction compared with the median level in the vehicle control group. Bioequivalent doxycycline mediated a 41.3% reduction in Wolbachia after +4 day exposures. In comparison, low dose rifampicin (5 mg/kg qd) mediated a significantly higher 66.3% reduction over the same time period (1 way ANOVA F = 7.561, P = 0.002, Dunnett's multiple comparisons test P < 0.05 vs DOX; Fig. 2b) whilst high dose rifampicin (15 mg/kg qd) mediated a 76.0% median reduction (P < 0.01 vs DOX; Fig. 2b). Elevations in oral rifampicin dose were undertaken to evaluate whether >90% threshold level Wolbachia depletions in female filarial tissues could be achieved in a time frame of seven days dosing. Six to seven week-old B. malayi timed infections were targeted with an additional washout period of five weeks to allow for continued   Table 2. Pharmacokinetic parameters of doxycycline and rifampicin and after single dose and chronic dose administration.
depletion or recrudescence of the Wolbachia population to be evident, as previously reported 59,60 ) (Fig. 2c). Doxycycline, at human bioequivalent (100 mg/day) dose 58 , was administered for four or six weeks (the latter a clinical dose time frame proven to deliver >90% Wolbachia depletion in B. malayi 39 to compare with the efficacy  of increasing dosages of short-course rifampicin regimens. As expected for 'slow-killing' anti-Wolbachia macrofilaricides, there were no significant changes in B. malayi worm burdens in either doxycycline or rifampicin dosed mouse groups compared with vehicle controls (Table 3). Doxycycline mediated an 88.1% median reduction in Wolbachia after four week dosing and 99.76% median reduction after six week dosing. In comparison, low dose rifampicin (5 mg/kg qd x 7d) was sub-optimal and statistically inferior to doxycycline, with a 58.3% median reduction (Kruskal-Wallis test P < 0.0001, Dunn's multiple comparisons test, P < 0.0001 vs +42d DOX; Fig. 2d). Elevation of rifampicin dose to 15 mg/kg qd, 35 mg/kg qd or 25 mg/kg bid delivered 97.7%, 98.2% or 99.5% median Wolbachia reductions, respectively. These dose effects were statistically non-inferior to six-week doxycycline dosing. Rifampicin 25 mg/kg bid dosing for seven days displayed significantly superior anti-Wolbachia activity to four-week doxycycline (P < 0.0001) and 15 mg/kg qd or 25 mg/kg bid rifampicin were superior to 5 mg/kg rifampicin after seven day dosing (P < 0.05 and P < 0.0001, respectively; Fig. 2d). Examining the impact of anti-Wolbachia efficacy on mf release, four or six-week doxycycline mediated a complete absence of viable mf within the peritoneal infection site (5/5 or 4/4 mice mf negative vs 2/25 mf negative vehicle; Kruskal-Wallis test P < 0.0001, Dunn's multiple comparisons test, P < 0.01 or P < 0.05 vs vehicle; Fig. 2f). Low-dose rifampicin (5 mg/kg qd) did not significantly affect the peritoneal B. malayi mf burden compared with vehicle control levels (0/4 mice mf negative; Fig. 2e). In comparison, seven day dosing with 15 mg/kg qd rifampicin mediated a near complete absence of viable mf from the peritoneum of infected mice (11/12 mice mf negative; P < 0.0001 vs vehicle; Fig. 2e). Seven-day doses of rifampicin exceeding 15 mg/kg qd mediated total absence of viable mf (35 mg/kg qd: 4/4 mice mf negative P < 0.05 vs vehicle, 25 mg/kg bid: 9/9 mice mf negative P < 0.001 vs vehicle; Fig. 2e).
Based on threshold effects of elevated dose rifampicin achieving >90% Wolbachia depletions from Brugia adult female tissues following seven day dosing, we tested efficacy against Onchocerca male worms in a novel SCID mouse model of onchocerciasis 56 Rifampicin dosed at 35 mg/kg qd for seven or 14 days was compared with human bioequivalent doxycycline dosed for 28 days (a minimum clinical dose/duration determined to deplete >90% Wolbachia from adult O. volvulus tissues leading to macrofilaricidal activity 50 , (Fig. 2f). Male Onchocerca worm burdens were not affected by treatment compared with vehicle controls (Table 3). Bioequivalent doxycycline mediated 99.6% median reduction in Wolbachia from male Onchocerca tissues following 28 days dosing (Fig. 2g). Seven-day dosing of rifampicin did not mediate any depletion in Wolbachia following washout period (0% median reduction) and was thus statistically inferior to doxycycline control (Kruskal-Wallis test P = 0.0013, Dunn's multiple comparisons test, P < 0.001 vs DOX; Fig. 2g). In comparison, 14 day dosing of rifampicin mediated 96.9% median reductions in Onchocerca Wolbachia, a level of efficacy that was statistically non-inferior to doxycycline dosed for 28 days (Fig. 2g).
PK-PD modelling. The empirical PK and pharmacological anti-Wolbachia efficacy measurements of doxycycline and rifampicin were used to construct a PK-PD model (described in the methods section). An in vivo IC 50 value was calculated based on the model for rifampicin (65.2 µg/ml), which was ~16-fold more potent than that calculated for doxycycline (1059 µg/ml; Table 1). IC 50 values derived from the PK-PD model were 100,000 to 200,000 fold higher than those observed in vitro; however the ratio of calculated in vivo IC 50 between rifampicin and doxycycline was aligned to the ratio empirically measured in vitro between the two drugs (~17-fold higher RIF vs DOX, Table 1).

Mouse -Human Bridging analysis.
To compare rifampicin exposures observed in the SCID mouse model in comparison to those achieved in standard human clinical dose (600 mg), the overall exposure of rifampicin has been estimated in humans based on literature data (Fig. 3a). From this we determined the exposure in an average population of 70 kg humans after receiving 600 mg dose of rifampicin is significantly lower than the  optimal exposure in mice that results in >90% Wolbachia elimination in 7 days or less (this exposure is defined as the 24 hour steady state AUC of the 15 mg/kg rifampicin dose in mice which is 141.5 mg/hr/L). The human dose that would achieve this optimal mouse exposure was then determined via multiple Monte-Carlo simulations (3000 subjects per dose) at different doses from 5 mg/kg up to 45 mg/kg where the percentage of humans predicted to achieve the desired AUC at each dose has been recorded (Fig. 3b). The PK predictions of higher doses of Rifampicin agree with published results where the predicted C max and AUC were equivalent to observed PK in humans at elevated doses in clinical studies 61 . This would indicate that our dosing suggestion will achieve the desired exposure based on clinical PK data. From those simulations, it is predicted that a minimum dose of 30-40 mg/kg is needed to achieve 'curative' exposure levels in human filariasis patients which also agrees with high dose RIF clinical studies 61 that have shown a dose of 30 mg/kg or higher to achieve a steady-state AUC of 141.5 mg/hr/L in the vast majority of patients

Discussion
Targeting Wolbachia with antibiotics to deliver curative outcomes for filariasis is a promising drug development strategy. The pharmacopeia of registered antibiotics present 'low hanging fruit' with the potential for rapid re-purposing 62,63 . Further, the 'slow kill' effect post-Wolbachia depletion, manifest first on circulating or skin mf and then adult worms, avoids the problems of inflammatory adverse reactions which are associated with fast-killing direct filaricidal agents 3,33,64-66 . Indeed, this promotes anti-Wolbachia therapies as a safe drug development strategy for to administer to L. loa co-endemic target populations 67 who are at risk of severe neurological adverse reactions to rapid acting filaricides and thus provides a potential solution to problem areas of Central Africa toward an end game of onchocerciasis elimination 68 . Clinical trials with doxycycline have shown that (optimal exposure is defined as the median total 24 hour steady state AUC in mice receiving daily 15 mg/kg dose). depletion of Wolbachia from filarial nematodes can deliver significant macrofilaricidal activity when administered for 4 or more weeks 38,[40][41][42]51 . However, the long period of administration and contraindications in children and pregnant women present logistical challenges to widespread scale-up in resource poor settings of Sub-Saharan Africa. Whilst we have recently identified minocycline as a superior anti-Wolbachia agent to doxycycline in the B. malayi SCID mouse model 58 as part of the same tetracycline class, the same contraindications would apply. The 'holy grail' for an anti-Wolbachia macrofilaricide is thus demonstrable activity in administration times of a minimum of 2 weeks (ideally 1 week) and a safety profile for administration in loiasis co-infections, in children and in pregnant women 34,63 .
We use here a validated screening system of in vitro cell assays and in vivo preclinical mouse models of filariasis 56,57 to test the efficacy of dose elevations of rifampicin and have identified a dose-dependent activity which has the potential to achieve Wolbachia depletions predictive of cure when administered for 1 week against the human lymphatic filaria B. malayi or 2 weeks against O. ochengi, the closest phylogenetic relative of O. volvulus.
We determine that rifampicin is >15 fold more potent than doxycycline in vivo. Rifampicin exhibited significantly higher exposure per mg dose when compared to doxycycline. Also PK-PD modelling showed that rifampicin is more potent in reducing Wolbachia in vivo when exposure levels are matched to doxycycline. This superiority likely reflects the inherently higher (>16 fold) intrinsic potency of rifampicin against Wolbachia that is observed in vitro compared with doxycycline.
Corroborating rifampicin superiority versus doxycycline as an anti-Wolbachia agent, it has previously been reported, in mice experimentally infected with the rodent filaria, Litomosoides sigmodontis, that 25 mg/kg bid dosages of rifampicin result in a more profound decrease in Wolbachia levels in adult worms when compared to a similar dose of doxycycline 48 . Also, in a pilot study using the related, registered rifamycin, rifapentine, >90% depletion of Wolbachia was measured in O. ochengi males implanted into SCID mice, following two weeks 25 mg/ kg bid dosing 56 . However, clinical studies in humans have shown that rifampicin administered for two or four weeks has inferior activity to six-week doxycycline in onchocerciasis 69,70 and neither is combining rifampicin and doxycycline for two weeks sufficient to achieve significant macrofilaricidal activity 54 .
Our PK-PD model offers a rational explanation for the discrepancy in the activity of rifampicin in mice and humans, where doxycycline activity is similar across mouse model and human clinical studies, yet rifampicin seems to be dramatically more active in the mouse than in humans. We identify effective dosages in the mouse exceed the exposure of rifampicin achieved in humans when the drug is administered at 'standard dose' (~10 mg/ kg qd or 600 mg). Indeed, exploring dose ranges more in line with standard human dose exposures, (5 mg/kg qd) rifampicin in the mouse is insufficient to achieve >90% Wolbachia depletion in 1 week against B. malayi.
The currently established 600 mg dose of rifampicin has not been predicated on PK-PD analysis but rather has been chosen based on drug cost, due to rifampicin semi-synthetic production in the 1970 s when the dosage was established 71 . However, recently the cost of rifampicin production has dramatically decreased and dose escalation studies have been initiated for TB patients. Higher doses of rifampicin (up to 35 mg/kg) have recently been proven to be safe in a clinical study 61 . The same study undertook human PK analysis and has shown that rifampicin exposure increases as the dose is increased. From this data we predict that the target exposure level required to deplete >90% Wolbachia from target filarial species in 1-2 weeks to ultimately deliver short-course curative efficacy against filariasis is achievable in humans when a dose of 30-35 mg/kg is administered. Importantly, clinical data supports that for doses of up to 35 mg/kg, given for periods over 2 weeks, no serious side effects are observed and that grade 1 and grade 2 side effects are randomly distributed across low and higher doses indicating no increased risk of side effects with elevated doses of rifampicin. A recent additional study where a dose of 35 mg/kg of RIF was administered to TB patients has also reported similar conclusions in relation to safety 72 .
Because elevated rifampicin dosages for filariasis cure are predicted in a 1-2 week dose exposure time frame, this is unlikely to result in resistant strains of M tuberculosis (TB), as there is no evidence for rifampicin resistance occurrence when administered for short periods of time 73,74 .
Rifampicin has a number of drug-drug interactions of clinical significance that have been previously reported 75 . Those interactions can result in the reduction of exposure of other drugs that are taken concomitantly with Rifampicin, and doses might need to be altered, especially for patients who are on HIV medication.

Conclusions
Using PK-PD modelling of drug activity in preclinical filarial mouse models, we show that a clinically safe dose of rifampicin can be administered for 1-2 week short courses to elicit >90% reductions in Wolbachia, predictive of the macrofilaricidal activity observed when Wolbachia is targeted by doxycycline, administered for 4-6 weeks. This treatment regimen would be compatible for use in children and during pregnancy and, because of the shortened duration of administration required, would be more readily deliverable by health care systems in resource-poor community settings. A 35 mg/kg dose has been proven as safe over a period of 2 weeks, and it has previously been shown that such short durations of treatment will not produce resistant strains of M. tuberculosis. We recommend that both a 35 mg/kg 1 and 2 week dose of rifampicin be urgently examined in lymphatic filariasis and onchocerciasis randomised controlled clinical trials to test whether such short-course treatment regimens are indeed sufficient to elicit equivalent curative activities to long course doxycycline.

Methods
Mice. Six  UK, or the University of Buea Animal Ethics Review Board, Cameroon. Studies were conducted in accordance with Home Office legislation (UK) and matching welfare standards were applied in Cameroon.
In vitro potency studies. The anti-Wolbachia potency of doxycycline and rifampicin for use in the PK-PD model was determined in vitro, utilising the routine A·WOL screening assay as described previously 57 . In brief the mosquito (Aedes albopictus) derived cell line C6/36 (ATCC number CRL-1660), stably infected with Wolbachia pipientis (wAlbB) (C6/36 wAlbB) was incubated with the relevant drugs in a concentration range in order to determine a dose response. The drugs were incubated for 7 days with 2,000 cells per well on a 384 well plate (CellCarrier-384 Ultra, PerkinElmer) in Leibovitz media (Life Technologies ™ ) supplemented with 20% foetal bovine serum (FBS, Fisher Scientific), 2% tryptose phosphate broth (Sigma-Aldrich) and 1% non-essential amino acids (Sigma-Aldrich). The end-point read out utilised DNA staining of both the host cell nuclei and intracellular Wolbachia (SYTO ® 11) combined with a high content imaging system (Operetta ® , PerkinElmer) and analysed using the associated Harmony ® software through a cytoplasm texture analysis.
Brugia malayi experimental infections. The Brugia malayi lifecycle was maintained through mosquitoes and susceptible Meriones gerbils at LSTM. To generate infective B. malayi larvae for infections, female adult Aedes aegypti mosquitoes were fed with microfilariae collected from infected gerbils by catheterisation, as described previously 76 , that were mixed with human blood. Mosquitoes were fed through an artificial membrane feeder (Hemotek ® ). Blood-fed mosquitoes were reared for 14 days to allow for development to the L3 stage. L3 were then collected from infected mosquitoes by crushing and concentrated using a Baermann's apparatus and RPMI medium. 100 motile L3 were collected and then injected into mice via the intra-peritoneal route. Efficiency of inoculations was confirmed by needle washout.
Onchocerca ochengi surgical implantations. Viable male Onchocerca ochengi were aseptically isolated from naturally parasitized cattle as described previously 56 . Between 10-11 male Onchocerca were surgically implanted into the peritoneal cavity of CB.17(BALB/c) SCID mice under anaesthesia as described previously 56 . Drug treatments. Six weeks after B. malayi experimental infection or 3 days post O. ochengi surgical implantation, SCID mice received 100 µL compound via oral gavage at variable doses and for variable treatment lengths as described and indicated in further detail in the main text. The drug doses and routes used were: Doxycycline (25 mg/kg qd or bid po and rifampicin (1.25 mg/kg-25 mg/kg qd or bid po). Doxycycline was dissolved in water, while rifampicin was dissolved in 55% polyethylene glycol 300; 25% propylene glycol; 20% water. All drugs and vehicle reagents were purchased from Sigma Aldrich.

Parasitological readouts. Seven or twelve weeks after B. malayi experimental infection or 38 days after
O. ochengi surgical implantation, mice were necropsied. Adult filariae and released B. malayi mf were recovered by a combination of peritoneal washings using wash media (RPMI containing P/S) and subsequent dissection of abdominal cavities. To quantify B. malayi mf, samples were centrifuged at 1200 rpm for 5 min. Excess supernatants were removed and the remaining volume quantified. Three 20 µL volumes were then enumerated for mf by microscopy, scoring as motile or stretched immotile. Adult filariae were observed for motility, washed in cold PBS and parasite stages enumerated. A minimum of ten B. malayi females and five O. ochengi males per treatment group of 3-5 mice were collected into individual Eppendorf tubes to be processed for qPCR. DNA extraction and PCR quantification of Wolbachia. DNA was extracted from worm samples using a DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer's instructions. Levels of Bm Wolbachia surface protein (wsp) gene copy numbers were quantified using qPCR as previously described 77 . Levels of Onchocerca Wolbachia wsp and filarial gst gene copy numbers were similarly quantified as previously described 56 . PK Studies. To establish the pharmacokinetic profiles of rifampicin and doxycycline, rich pharmacokinetic studies were performed in uninfected male SCID mice (weight 24-28 g). Doxycycline (25 mg/kg bid) or rifampicin (5 mg/kg qd or 25 mg/kg bid) were administered orally for up to seven days. For all PK studies, a total of 11 samples were collected at discrete time points between 0 and 24 hour post dose at days 1 and 7). Serial blood samples were collected at days 1 and 7 of dosing via the tail vein where a microincision was performed and 20 µL of blood collected using a pipette with a pre-heparinised tip. Blood samples were directly lysed with 40 µL of ice cold ddH 2 O and then frozen at −80 °C until time of bioanalysis.
Bioanalysis. Rifampicin concentrations were determined by LCMS (liquid chromatography mass spectrometry) using an appropriate internal standard and were validated to internationally recognised acceptance criteria [FDA-guidelines]. Chromatographic separation was achieved using a gradient programme. The UHPLC (ultrahigh pressure liquid chromatography) system was interfaced with a triple-quadruple TSQ Quantum Access mass spectrometer (Thermo Scientific, Hemel Hempstead, UK) with a heated-electrospray ionization (H-ESI) source. An E2M28 rotary vacuum pump (Edwards High Vacuum International, West Sussex, UK), an NM30LA nitrogen generator (Peak Scientific, Renfrewshire, UK) and 99% pure argon gas (10 L, BIP10, Air Products, Liverpool, UK) were used. Blood samples (20 μl) containing rifampicin were extracted with a mixture of acetonitrile and methanol (80:20 v/v). The subsequent mix was then filtered through 96 well filter plate (Millipore, UK). The resultant filtrate was then transferred to a 96 deep well plate containing 40 µl of (10 mg/ml) ascorbic acid, 10 µl of this resultant mixture was then injected into the LC-MS/MS. The assay was linear in the range 10-10,000 ng/ml for rifampicin.

PK-PD Modelling.
A PK-PD model as described in equations 1 and 2 has been developed and used to fit parasitological read-outs from experiments in infected mice. The pharmacological output for all PK/PD analyses Scientific REPORTS | _#####################_ | DOI:10.1038/s41598-017-00322-5 was Wolbachia counts as estimated in qPCR experiments in the quantitation of wsp DNA levels. Pmetrics ® 78 was used in modelling PK/PD data in all experiments. The following differential equations were used: For the PK component: Where C p is the drug concentration in blood at any given time, F is the fraction available for oral absorption, k a is the rate of absorption from gut to blood, V is the volume of distribution and k el is the rate of elimination from the systemic circulation. For the PD component, a relationship was built to describe the effect of the drug upon Wolbachia levels using a dynamic E max model: growth kill p p 1 1 50 X 1 is the observed Wolbachia levels as estimated by qPCR, k growth is the rate of bacterial growth which was set at 0 due to the absence of any evidence of bacterial growth beyond the time of treatment and no evidence of recrudescence within the time frame of the experiment. K kill is the maximum possible rate of kill of bacteria and has been fixed to the in vitro kill rate that could be achieved at the maximum concentration of drug. IC 50 is the blood concentration required to achieve 50% of the maximal kill rate of Wolbachia in the adult worm in the mouse.
Statistics. PK parameters between doxycycline and rifampicin were compared by Mann-Whitney test.
Percentage Wolbachia reductions in filariae following in vivo treatments were calculated from median vehicle control levels derived from the same experimental infection and screen. Where repeat experiment data (Wolbachia loads normalised to control, worm enumerations or viable mf enumerations per mouse) were available, data was pooled. Grouped continuous variables were tested for normal distribution by D' Agostino & Pearson omnibus normality tests. Continuous variables failing normal distribution tests were Log 10 transformed and re-tested. Continuous variables satisfying the assumptions of normal distribution were examined by 1 way ANOVA with Dunnett's multiple tests post-hoc. Variables not satisfying the assumption of normality were compared by Kruskal-Wallis Test with Dunn's Multiple Tests, post-hoc. Significance levels are indicated P < 0.05*, P < 0.01** P < 0.001*** P < 0.0001****. All statistics were undertaken using GraphPad Prism v6 software.