Preclinical pharmacokinetics and tissue distribution of a polyhexamethylene guanidine derivative after ocular mucosal administration

Abstract

Antibiotic resistance is a critical medical issue. Antiseptics, which play a key role in treating and preventing eye infections and inflammations, have the potential to induce resistance in pathogenic microorganisms despite their broad antimicrobial spectrum. When introducing new active substances into ophthalmic treatments, it is crucial to assess the risk of side effects caused by the systemic action of the active compound. The succinic salt of polyhexamethylene guanidine derivative (ss-PHMGd) has shown high in vitro activity. This study aimed to evaluate the systemic effects of ss-PHMGd-based eye drops after ocular instillation in a preclinical model. Simple mathematical models were used to calculate pharmacokinetic parameters during instillation in two types of laboratory animals: guinea pigs and chinchilla rabbits. Groups received intravenous injections and ocular treatments. Tritium-labeled ss-PHMGd was used to quantify its concentration in organs and tissues. To test the linearity of the parameter relationships, data from three different doses were compared. Renal excretion was studied by quantifying ss-PHMGd concentrations in urine using radiometric methods. The results showed that ss-PHMGd-based eye drops present a low risk of systemic side effects and exhibit minimal penetration into other organs after instillation. No species-specific differences were found in the ADME parameters of the preclinical models (the guinea pig-to-chinchilla rabbit absorption ratio was 0.7). The AUC-D parameters exhibited a linear relationship for doses ranging from 0.5 of therapeutic dose (TD) to 5TD in guinea pigs. Renal excretion analysis revealed that 5.3% of the administered dose was excreted through the kidneys, indicating significant metabolic transformation or excretion via other pathways. This study enhances the understanding of the pharmacokinetics and safety of polyguanidines, with implications for toxicology and risk assessment.

Introduction

Acquired antibiotic resistance is a significant concern including ophthalmology. Gram-positive Staphylococcus spp. and gram-negative bacteria, such as P. aeruginosa, E. coli, and K. pneumoniae, are frequently identified in infected patients [1]. Resistant gram-negative pathogens, particularly P. aeruginosa, have raised alarms, with the Centers for Disease Control and Prevention issuing advisories on resistant strains [2]. Fluoroquinolones and aminoglycosides are commonly used for empirical therapy, but growing resistance among pathogens necessitates the search for alternative treatments [3-7].

The World Health Organization’s 2015 global action plan highlighted the challenge of antimicrobial resistance, stressing the need for more effective treatments [8, 9]. Antiseptic agents, including those used in ophthalmology, have become crucial in managing eye infections. Despite their broad antimicrobial spectrum, antimicrobial resistance in these agents is emerging, especially against agents like picloxydine dihydrochloride and benzyl dimethyl[3-myrystoylaminopropyl] ammonium chloride monohydrate [10-13]. Povidone-iodine and chlorhexidine are common antiseptics, but they may cause significant ocular irritation and toxicity, leading to concerns about their safety in ophthalmic use [14-21].

Polyhexamethyleneguanidine derivatives (PHMGd), particularly the succinic acid salt (ss-PHMGd), show promise in combating resistant pathogens. ss-PHMGd has demonstrated antimicrobial and biofilm-disrupting properties, with potential advantages due to the biosafety of succinic acid [22-25]. However, ocular antiseptics like ss-PHMGd may pose systemic risks, as some active substances can be absorbed through the eye and enter the bloodstream, potentially causing adverse effects in other organs. Given the high pulmonary toxicity of polyguanidines, it is essential to assess their systemic exposure and safety [26]. This study aims to evaluate the systemic exposure of ss-PHMGd-based eye drops following ocular instillation (OI), contributing to the understanding of the pharmacokinetics and safety of this compound in the context of human health.

Materials and Methods

Active pharmaceutical ingredient

Characterization

The active pharmaceutical ingredient in the investigated dosage form was ss-PHMGd. As previously noted, this compound shares structural similarities with PHMG, yet exhibits significant differences. Notably, in contrast to the linear polymeric structure of PHMG, ss-PHMGd features a branched architecture, quantitatively described by its degree of branching (z) [27-29].

Synthesis of active pharmaceutical ingredient

One part of a 50% aqueous solution of PHMGd hydrochloride (Pharma-Pokrov LLC, Russia) was mixed with ten parts of a solution containing 2–4 molar equivalents of alkali (potassium hydroxide (Component-Reactive LLC, Russia)) dissolved in 96% ethanol (Component-Reactive LLC, Russia). The high alkali-to-PHMGd ratio enabled a reduction of the chlorine content in the PHMGd base to 0.2–0.5%, corresponding to a degree of substitution above 95%. Due to the high solubility of the alkali in the water-ethanol medium, this process led to an increase in the ash content of the product. To mitigate this, carbon dioxide was bubbled through the solution, converting the excess alkali into potassium bicarbonate, which is poorly soluble in the alcoholic medium. Concurrently, the PHMGd base was transformed into PHMGd hydrocarbonate, which remained dissolved in the solution. The resulting solution was purified by filtering off the precipitated potassium bicarbonate. To obtain ss-PHMGd, an equimolar amount of succinic acid was added to the PHMGd hydrocarbonate solution, followed by solvent evaporation using a rotary evaporator. The resulting ss-PHMGd had a number-average molecular weight (Mn) of 800 Da and z≈0.4 [28, 30].

Radiolabeling of ss-PHMGd with tritium

For the quantitative determination of ss-PHMGd in the organs and tissues of experimental animals, a tritium-labeled compound (³H-ss-PHMGd) was used. Tritium labeling was achieved by thermal activation [31-33].

A 5 μL aliquot of ss-PHMGd solution (25 mg/mL) was applied to a glass reaction vessel, treated with 500 μL of 70% ethanol, and uniformly distributed. The solvent was removed via rotary evaporation at 20 °C to form a thin polymer film. The vessel was mounted into a stainless-steel high-vacuum system (down to 1 × 10⁻⁶ mbar; ILMVAC GmbH, Germany) and cooled to 77 K using liquid nitrogen. Gaseous tritium was introduced (5 × 10⁻³ mbar), and a tungsten filament aligned along the vessel axis was heated to 1900–2000 K for 10 s. This irradiation cycle was repeated five times. The labeled product was washed with 50% aqueous ethanol by repeated rotary evaporation (up to five times) to remove labile tritium.

Twelve samples (1.5 mg each) were irradiated. The final labeled product was dissolved in 3 mL of 50% ethanol, divided into three vials, and dried using a Vortex-Evaporator (Thermo Fisher Scientific, USA), yielding three portions of ³H-ss-PHMGd (0.5 mg each, 14.1 mCi/mg; total activity 21.2 mCi). Thin-layer chromatography (TLC) analysis was performed on silica gel 60 plates (Merck KGaA, Germany) using water:isopropanol (10%) with 10% KCl as mobile phase. Visualization was done with ninhydrin, and radioactivity was measured by liquid scintillation counting (Wallac 1409, PerkinElmer, USA). The labeled compound co-migrated with the unlabeled reference.

Dosage forms

Intravenous Administration (Chinchilla rabbits)

For intravenous (i.v.) injection in Chinchilla rabbits, a sterile formulation containing ³H-ss-PHMGd at a final concentration of 0.07 mg/mL in physiological saline was prepared. The formulation was obtained by diluting a 2.5 mg/mL stock solution of ³H-ss-PHMGd (in water for injection) 35.5-fold with sterile saline.

Intravenous Administration (Guinea Pigs)

A mixed formulation containing both ³H-ss-PHMGd and non-labeled ss-PHMGd was prepared in sterile physiological saline at a final total concentration of 0.024 mg/mL. The preparation involved sequential dilution: the ³H-ss-PHMGd stock solution (2.5 mg/mL) was diluted 141.8-fold with sterile saline, followed by a 161.5-fold dilution of the ss-PHMGd stock solution (1.0 mg/mL in water for injection) with the previously prepared radioactive solution. The final volume was based on the number of animals. The 1.0 mg/mL ss-PHMGd solution was prepared fresh prior to formulation.

Ocular Instillation (Guinea Pigs)

For OI in guinea pigs, a 0.05% solution of ³H-ss-PHMGd and ss-PHMGd was prepared. Fresh stock solutions of ss-PHMGd (1.0 mg/mL in water for injection) and excipients were used. The excipient stock solution contained (per liter): NaCl – 4.15 g, hydroxypropyl methylcellulose – 4.84 g, KH₂PO₄ – 7.34 g, Na₂HPO₄·12H₂O – 20.28 g. The formulation was prepared by mixing the ³H-ss-PHMGd solution (2.5 mg/mL), the 1.0 mg/mL ss-PHMGd solution, and the excipient solution in a ratio of 1.2 : 1 : 5.7, respectively. The final pH of the recipe was 7.2.

Ocular Instillation (Chinchilla rabbits)

For Chinchilla rabbits, a 0.05% solution of ³H-ss-PHMGd was prepared. The fresh excipient stock solution contained (per liter): NaCl – 3.75 g, hydroxypropyl methylcellulose – 4.38 g, KH₂PO₄ – 6.64 g, Na₂HPO₄·12H₂O – 18.34 g. The formulation was prepared by mixing the ³H-ss-PHMGd solution (2.5 mg/mL) with the excipient solution in a 1 : 4 ratio. The final pH of the recipe was 7.2.

Animals, housing, group distribution and drug administration

Outbred male guinea pigs (2.2–2.8 months, 350–400 g; n = 300 + 30 reserve) and female Chinchilla rabbits (2.0–2.5 months, 2.0–2.5 kg; n = 12 + 2 reserve) were obtained from the Scientific Center for Biomedical Technologies, Federal Medical and Biological Agency of Russia (Andreevka facility).

Animals were housed in species-specific rooms under standard conditions (temperature 18–20 °C, humidity 30–70%, 12 h light/dark cycle, ≥11 air changes/hour). Guinea pigs were kept in polycarbonate cages (2 per cage, LabProdex®, USA); rabbits were housed individually in stainless steel cages (Techniplast®, Italy). For urine collection, guinea pigs were temporarily placed in metabolic cages (Techniplast®, Italy).

Animals had ad libitum access to filtered tap water and standard laboratory diet (PK-120-1; Laboratorsnab LLC, Russia). Bedding (wood shavings) and feed were regularly tested for microbial contamination.

Acclimatization lasted 21 days for guinea pigs and 14 days for Chinchilla rabbits. Animals were monitored twice daily. Grouping was based on study design and body weight (± 10% variation within groups). Identification was done via permanent ear markings and cage labeling. Reserve animals were housed separately.

Animals were divided into groups with less than 10% weight variation (5-6 animals per group) [34-37]. The distribution of animals into groups depending on the method of drug administration is presented in Table 1.

Table 1.

Distribution of animals into groups depending on the method of drug administration.

Group	Animals	Subject numbers	Dose	Route of administration	Volume of instillation, ml
I	Guinea pigs	n=50	TD1	i.v.4	0.2
II	Guinea pigs	n=50	TD	OI5	0.005
III	Chinchilla rabbits	n=60	TD	i.v.	0.2
IV	Chinchilla rabbits	n=60	TD	OI	0.011
V	Guinea pigs	n=50	0.5TD	OI	0.0025
VI	Guinea pigs	n=50	5TD	OI	0.0025
VII	Guinea pigs	n=50	TD2	OI	0.005
VIII	Guinea pigs	n=50	TD3	OI	0.005
IX	Guinea pigs	n=5	TD	OI	0.005

¹- therapeutic dose;

²- twice with an interval of 24 hours;

³- three times with an interval of 24 hours;

⁴- intravenous;

⁵- ocular instillation.

I.v. administration of the drug to guinea pigs and Chinchilla rabbits was performed using disposable insulin syringes into the subcutaneous vein of the leg and the marginal ear vein respectively.

The individual volume of the administered formulation for each group of animals was adjusted based on the average weight of the animals in the group. The difference in ocular instillation volumes between guinea pigs (5 μL) and Chinchilla rabbits (11 μL) was based on species-specific anatomical features, including conjunctival sac capacity [38–40], and aligned with the Guide for Working with Laboratory Animals in Preclinical (Non-Clinical) Studies in the EAEU [41]. The drug concentration was identical for both species; thus, volumes were selected to ensure adequate dosing without exceeding ocular tolerability. In contrast, intravenous dosing was administered as a fixed volume (0.2 mL per animal), adjusted by body weight, and required no species-specific modification.

The daily therapeutic dose (TD) was 0.012 mg/kg and 0.007 mg/kg for guinea pigs and Chinchilla rabbits respectively [36]. Taking into account the specific activity of ³H-ss-PHMGd, its additions to the formulations according to the daily TD were 0.0089 mg/kg (74%) for guinea pigs and 0.007 mg/kg (100%) for Chinchilla rabbits, which was done based on interspecies differences in ocular uptake and tissue distribution in animals, where 74% is taken from the reference TD value established in the Chinchilla rabbits model.

All procedures adhered to international ethical guidelines [42-45] and were approved by the Local Ethics Committee of the Research Center for “Toxicology and Hygienic Regulation of Biopreparations” at Federal Medical and Biological Agency of Russia. The experiment was also approved by The Local Bioethics Advisory Commission of The Research Center for ‘Toxicology and Hygienic Regulation of Biopreparations; at Federal Medical and Biological Agency of Russia of Russia (Permission number for animal experiments: 009/1017).

Alkaline mineralization

For mineralization, 2 ml of 3 M potassium hydroxide was added per 50-200 mg of biological sample in test tubes, which were then heated in a boiling water bath for 40 minutes until fully dissolved. For whole blood or blood-rich samples, 0.05 ml of 50% hydrogen peroxide was also added. After cooling, 0.2 ml aliquots of the mineralized samples were transferred to scintillation vials with 10 ml of Optiphase HiSafe scintillation fluid (PerkinElmer, USA). The ³H-ss-PHMGd content was quantified using a Triathler Multilabel Tester (PerkinElmer, USA) based on scintillation levels, following regression analysis. Calibration curves were constructed using the scintillation response to labeled preparation amounts in mineralized blood, major organ tissues, and urine samples from intact animals [46].

In vivo systemic pharmacokinetic studies

Pharmacokinetic (PK) parameters were assessed, including elimination half-life (t_1/2), total clearance (CL), steady-state volume of distribution (V_ss), apparent volume of distribution (V_d), area under the concentration vs time curve (AUC∞), area under the PK curve (AUMC), mean residence time (MRT), maximum concentration (C_max), and time to reach C_max (T_max).

PK studies were performed in multiple groups according Table 1. Group I/III: blood samples was collected at 1, 2, 5, 10, 30 minutes, 1, 2, 5, 24 hours, and 3 days post-administration. Group II/IV: blood samples were taken at 10, 20, 30 minutes, 1, 2, 4, 24 hours, 3, 5, and 6 days. Experiments were conducted with guinea pigs (5 animals per time point) and Chinchilla rabbits (6 animals per time point). Blood samples were collected from the ear vein at 10 different time points, and tissue samples were collected for analysis after euthanasia [47].

Assessment of dose proportionality

To evaluate the dose proportionality of systemic exposure to ³H-ss-PHMGd, three dose levels were administered to outbred guinea pigs via ocular instillation: 0.0044 mg/kg (0.5TD), 0.0089 mg/kg (1TD), and 0.044 mg/kg (5TD), corresponding to Groups V and VI (Table 1). Blood samples were collected at 10, 20, 30 minutes, and 1, 2, 4, 24 hours, and on days 3, 5, and 6 post-dose.

Dose proportionality was assessed by weighted linear regression of AUC∞ versus dose, using the model: AUC∞=a+b×D [48-49].

Weights (w_j) were defined as the inverse of variance at each dose level w_j = S_j^-2 to account for heteroscedasticity.

The weighted mean of AUC∞ was calculated using Equation (1), with m=3 (number of doses):

$${\overline{AUC}}_w=\frac{{{\displaystyle \sum }}_{j=1}^m{w}_j{\overline{AUC}}_j}{{{\displaystyle \sum }}_{j=1}^m{w}_j}$$ (1)

Regression coefficients “bw” and “aw” and their variances were estimated by formulas (2) and (3):

$$D\left(b_w\right)=\frac{S_w^2}{\sum w_j\left(x_j-{\overline{x}}_w\right)}$$ (2)

$$D\left(a_w\right)={\overline{x}}_w^{2}D\left(b_w\right)$$ (3)

The residual variance was calculated by formula (4):

$$S_w^2=\frac{1}{m-2}\left[{{\displaystyle \sum }}_{j=1}^m{w}_j{\left(y_j-b_w{x}_j-a_w\right)}^2\right]$$ (4)

Dose proportionality was concluded if the intercept “aw” was not significantly different from zero.

Dose proportionality of AUC∞ also was assessed using the power model approach, which is recommended for pharmacokinetic dose-proportionality analysis. The model is described by formula (5):

$$\log \left(AU{C}_{\infty }\right)=\alpha +\beta \times \log (TD)$$ (5)

where α is the intercept, the estimated slope (β) is the dose-proportionality coefficient, and Dose represents the administered dose (mg/kg). The AUC∞ values and doses were logarithmically transformed, and linear regression was performed to estimate β and its 90% confidence interval (CI). Dose proportionality was concluded if the 90% CI for β included 1. Statistical analysis was performed using Python 3.10 (SciPy library).

In vivo tissue disposition studies

The accumulation of ³H-ss-PHMGd in the tissues of internal organs was studied according Table 1.

Blood samples were collected at 10, 20, 30 minutes, 1, 2, 4, 24 hours, 3, 5, and 6 days after the second instillation in group VII animals and after the third instillation in group VIII animals. Subsequently, the animals were euthanized, and autopsies were performed to examine tissue samples from the following organs: lungs, heart, liver, thymus, kidneys, spleen, skeletal muscles, and brain. The average PK parameters AUC∞ and C_max, calculated within the models, as well as the sample mean C_min (representing the concentrations of ³H-ss-PHMGd measured at 5-day intervals after each administration), were compared using the Newman–Keuls test (q). According to the Newman-Keuls test, the means were first sorted in ascending order and then compared in pairs by calculating the test statistic using formula (6), where ${\overline{x}}_A$ and ${\overline{x}}_B$ the means being compared (AUC∞, C_max, C_min), S2int is the within-group variance (i.e., averaged across the 3 groups in the case of three administrations), and n_A and n_B are the sample sizes of the groups.

$$q_{\text{critical}}=\frac{{\overline{x}}_A-{\overline{x}}_B}{\sqrt{\left(1/n_A+1/n_B\right)\times S_{\text{int}}^2/2}}$$ (6)

These values were compared with data obtained from the experiments. Data from group II animals were used for calculations in this experiment. Cumulation of ³H-ss-PHMGd in several organs was observed upon analysis of PK profiles after each OI and comparison of graphs depicting actual concentrations of ³H-ss-PHMGd with corresponding superposition of exponential graphs.

In vivo renal excretion study

Study of renal excretion of ss-PHMGd was conducted according Table 1.

Next parameters were investigated (value of excretion, value of excretion, t_1/2, maximal speed of excretion, common diuresis). Urine samples were collected on average twice daily with volume and collection time recorded as accumulation proceeded. The concentration of ³H-ss-PHMGd in urine samples was quantitatively determined using a radioisotopic method, after which the experiment was continued until the radioactivity of the samples reached background levels. The duration of the experiment was 7 days from the time of administration.

Statistical analysis

PK data and tissue disposition were analyzed using WINNONLIN NONLINEAR ESTIMATION PROGRAMM, Version 6.3.0.395, Core Version 16 Nov 2010. The Gauss-Newton algorithm in the Levenberg-Hartley version was used to select the parameters.

C_max and T_max were determined as averages. Absolute bioavailability (f_abs) was calculated as the ratio of AUC∞ between i.v. and OI groups (Table 1).

Dose proportionality analysis for Groups II, V, and VI followed [33]. Parameters like AUC∞, C_max, and C_min were compared using one-way ANOVA with post hoc Newman-Keuls test. Statistical significance was defined at p<0.05.

Renal excretion parameters were computed using the trapezoidal method with linear interpolation, and values were averaged.

Results and Discussion

Systemic pharmacokinetic studies

A semi-logarithmic plots of ³H-ss-PHMGd concentration (μg/ml) versus time in blood for both guinea pigs and rabbits following both administration routes was obtained (Figure 1). The primary PK parameters were calculated and summarized in Table 2.

Figure 1.

Figure 1. A semi-logarithmic plots of ³H-ss-PHMGd concentration (μg/ml) versus time in blood of experimental animals at both roots of administration: (a) guinea pigs with a single i.v. administration of TD (0.012 mg/kg); (b) guinea pigs with a single OI administration of TD (0.012 mg/kg); (c) Chinchilla rabbits with a single i.v. administration of TD (0.007 mg/kg); (d) Chinchilla rabbits with a single OI administration of TD (0.007 mg/kg); (e) guinea pigs with a single OI administration of 0.5TD (0.006 mg/kg); (f) guinea pigs with a single OI administration of 5TD (0.06 mg/kg); (g) guinea pigs with a double OI administration of TD (0.012 mg/kg); (h) guinea pigs with a triple OI administration of TD (0.012 mg/kg); (i) guinea pigs with a triple OI administrations of TD (0.012 mg/kg) at 24-hour intervals - modeled using the principle of exponential superposition; (k) guinea pigs with a triple OI administrations of TD (0.012 mg/kg) at 24-hour intervals in a real experiment.

i.v. – intravenously; OI – ocular instillation; x1 - single administration; x2 – double administration; x3 – triple administration; TD – therapeutic dose.

Table 2.

Average values of the main PK parameters of ³H-ss-PHMGd.

Average value ± SD1
Group	GP2, siv3	CR4, siv					GP, sOI5					CR, sOI
Organ	Blo6	Blo	Blo	Spleen	Lungs	Brain	Liver	Kid7	Sm8	Heart	Thy9	Blo
n	50	60	50	50	50	50	50	50	50	50	50	60
t1/2, min	1388±219.3	2356±435.9	1733±254.8	1840±311.0	2167±292.5	2200±281.6	1543±245.3	1698±307.3	1849±310.6	2294±312.0	1967±285.2	2743±348.4
AUC∞10, min×μg/ml	186.5±28.2	332.6±59.4	10.8±1.4	12.6±1.9	12.8±1.6	65.4±7.6	15.2±2.2	33.0±5.6	12.7±1.9	13.3±1.7	9.5±1.3	28.7±3.3
Tmax11, min	-	-	62.5 ± 8.0	77.5 ± 10.7	33.7 ± 7.4	94.3 ± 10.2	39.0 ± 7.1	21.3 ± 12.1	78.3 ± 10.7	32.6 ± 7.8	21.1 ± 11.1	80.7 ± 10.0
Cmax12, µg/g	-	-	0.0042 ± 0.0002	0.0046 ± 0.0002	0.004 ± 0.0001	0.020 ± 0.0008	0.0067 ± 0.0002	0.013 ± 0.0005	0.0046 ± 0.0002	0.0040 ± 0.0001	0.0033 ± 0.0001	0.007 ± 0.0003
CL13, l/h	0.0011±0.0002	0.0025±0.0004	-	-	-	-	-	-	-	-	-	-
Vss14, l	0.038±0.0010	0.14±0.0042	-	-	-	-	-	-	-	-	-	-
Vd15, l/kg	0.095±0.0025	0.07±0.0021	-	-	-	-	-	-	-	-	-	-
AUMC16, min×min×µg/ml	373326 ± 114984.4	1130562 ± 409263.4	-	-	-	-	-	-	-	-	-	-
MRT17, min	2002±316.3	3399±628.8	-	-	-	-	-	-	-	-	-	-

¹standard deviation;

²guinea pigs;

³single intravenously;

⁴Chinchilla rabbits;

⁵single ocular instillation;

⁶blood;

⁷kidney;

⁸skeletal muscles;

⁹thymus;

¹⁰area under the concentration vs time curve;

¹¹time taken to reach the maximum concentration;

¹²maximum concentration;

¹³total clearance;

¹⁴steady-state volume of distribution;

¹⁵apparent volume of distribution;

¹⁶area under the PK curve;

¹⁷mean residence time.

As it is shown in Table 2 when administered TD OI to guinea pigs t_1/2 was 1.25 times higher than the t_1/2 i.v. The longest t_1/2 for guinea pigs after OI was observed in heart, the least one in liver. The t_1/2 in the blood of Chinchilla rabbits after OI was 1.16 times higher than one after i.v. This is similar to the data about guinea pigs. The differences in t_1/2 values between OI and i.v. administration in both species are relatively modest (1.3-fold or less), and the AUC values are also comparable. The data indicate that following OI, the compound is absorbed sufficiently to produce systemic exposure levels similar to i.v. administration, suggesting a notable systemic bioavailability via the ocular route. Our findings indicate that OI results in a t_1/2 that is, on average, 1.2 times longer compared to i.v. administration. Perhaps this extended t_1/2 may be attributed to the slower release from ocular tissues and prolonged systemic circulation. This observation is particularly relevant for ophthalmological applications, where a prolonged effect could significantly enhance therapeutic efficacy and reduce the frequency of administrations. Additionally, this could be beneficial in the comprehensive treatment of bacterial infections, as the sustained drug presence may reduce the need for frequent administration of other systemic antibiotics and enhance the synergistic effect. Therefore, further studies on the long-term safety and efficacy of chronic administration are warranted.

The highest T_max for guinea pigs after OI was in brain, the lowest T_max was in thymus. A similar dependence was observed for these organs of the guinea pigs in AUC∞ parameter after OI. According AUC∞ data significant accumulation after OI also detected in kidneys and heart (Table 2). Tissue concentration-time curves for major organs of distribution (brain, kidneys and heart) present in Figure 2.

Figure 2.

Tissue concentration versus time curves for the main organs where accumulation was observed (brain, kidneys, heart, respectively): (a) guinea pigs with a single OI administration of TD (0.012 mg/kg), object: brain; (b) guinea pigs with a triple OI administration of TD (0.012 mg/kg), object: brain; (c) guinea pigs with a triple OI administration of TD (0.012 mg/kg) at 24-hour intervals - modeled using the principle of exponential superposition, object: brain; (d) guinea pigs with a triple OI administration of TD (0.012 mg/kg) at 24-hour intervals in a real experiment, object: brain; (e) guinea pigs with a single OI administration of TD (0.012 mg/kg), object: kidneys; (f) guinea pigs with a triple OI administration of TD (0.012 mg/kg), object: kidneys; (g) guinea pigs with a triple OI administration of TD (0.012 mg/kg) at 24-hour intervals - modeled using the principle of exponential superposition, object: kidneys; (h) guinea pigs with a triple OI administration of TD (0.012 mg/kg) at 24-hour intervals in a real experiment, object: kidneys; (i) guinea pigs with a single OI administration of TD (0.012 mg/kg), object: heart; (k) guinea pigs with a triple OI administration of TD (0.012 mg/kg), object: heart; (l) guinea pigs with a triple OI administration of TD (0.012 mg/kg) at 24-hour intervals - modeled using the principle of exponential superposition, object: heart; (m) guinea pigs with a triple OI administration of TD (0.012 mg/kg) at 24-hour intervals in a real experiment, object: heart.

OI – ocular instillation; x1 - single administration; x3 – triple administration; TD – therapeutic dose.

It is well known that the plasma membrane allows the passage of only molecules within a specific range of molecular size, polarity, and charge. One study reports a guanidine-containing molecular transporter capable of achieving this [50]. In general, cationic antimicrobial peptides are attracting significant attention due to their broad potential in treating inflammatory diseases, particularly those affecting the brain [51]. It is important to note that peptide–drug conjugates represent an effective method for the selective delivery of drugs in tumor diseases, and such conjugations are often carried out in solution phase using guanidinium salts as coupling agents [52]. The observed presence of ss-PHMGd in brain tissue after OI may indicate the possibility of the blood–brain barrier (BBB) crossing, although the exact mechanism remains unclear and requires further study. This observation is consistent with previously reported data for cationic polymers such as polyethyleneimine (PEI), which have demonstrated BBB penetration in some models, likely through adsorption-mediated transcytosis or paracellular pathways [53,54]. For example, Zhao et al. (2020) reported that a PEI-based nanoplatform accumulated in glioma tissue mechanisms of adsorption-mediated following systemic administration, enabling imaging and drug delivery into brain tissue [53]. While this analogy suggests a potential mechanism for ss-PHMGd, the current findings should be interpreted with caution, and additional studies are needed to confirm whether similar processes occur. In contrast, a study on male Alderley Park rats reported that after oral administration of radiolabeled polyaminopropylbiguanide (PHMB)—a compound related to ss-PHMGd—the highest radioactivity occurred in adipose tissue, followed by kidneys and liver, without detectable brain accumulation, highlighting the influence of administration route [55–57]. Therefore, the relatively high AUC∞ for ss-PHMGd in brain tissue following OI should be viewed as a preliminary observation rather than conclusive evidence of BBB penetration. Future studies, including mechanistic experiments, are required to clarify this phenomenon.

Value f_abs for guinea pigs as a ratio between AUC∞ in blood after OI and the one after i.v. amounts to 5.8%. As a result of the refinement, the f_abs parameter amounted to 5.9% for guinea pigs (with the lower and upper boundary of 95% CI that equals 0.039 and 0.088, respectively). Value f_abs for Chinchilla rabbits was defined in a similar way and equaled 8.6% but the updated f_abs indicator equaled 8.9% (with the lower and upper boundary of 95% CI that equals 0.058 and 0.138, respectively). The resulting values are statistically indistinguishable, so it can be claimed that there is no species specificity.

The interspecies bioavailability coefficient, calculated as the ratio of f_abs in guinea pigs to that in Chinchilla rabbits, was 0.7 [39]. This difference may reflect species-specific anatomical and physiological variations influencing ocular absorption. For example, guinea pigs have a thinner corneal epithelium, smaller tear volume, and different blink rate compared to Chinchilla rabbits, factors that can affect drug retention and corneal penetration. Additionally, differences in conjunctival vascularization and enzymatic activity in ocular tissues may contribute to variability in first-pass metabolism or elimination from the ocular surface. Several studies highlight substantial anatomical and physiological differences between these species. For instance, under Schirmer Tear Test I (STT I), guinea pigs exhibited tear production of 3 mm/min, whereas Chinchilla rabbits demonstrated values almost three times lower. Similarly, under the Phenol Red Thread Test (PRTT), guinea pigs showed 21.2 mm/15 sec, while Chinchilla rabbits had values 1.5 times lower. Corneal characteristics also differed markedly: guinea pigs presented a corneal thickness of 6.64 g/mm² versus 10.84 g/mm² in Chinchilla rabbits. Central corneal thickness and endothelial cell density were 227.85 μm and 2352 cells/mm² in guinea pigs, compared to 340 μm and 3423 cells/mm² in Chinchilla rabbits [58]. Another study reported significant differences in intraocular pressure (IOP): 6.81 ± 1.41 mmHg in guinea pigs versus 17.38 ± 4.66 mmHg in male Chinchilla rabbits and 18.41 ± 3.21 mmHg in females [59–60]. These findings demonstrate that tear production, corneal morphology, and IOP differ considerably between the two species and may influence ocular absorption and PKs.

Values of f_abs for ss-PHMGd following OI were 5.9% in guinea pigs and 8.9% in Chinchilla rabbits, indicating limited systemic bioavailability compared to i.v. administration. Although the observed interspecies bioavailability ratio (0.7) suggests broadly comparable PK behavior, the potential for species-specific variation should not be disregarded. These differences should be considered when extrapolating preclinical ocular PK data to humans. Nevertheless, the overall similarity in systemic absorption profiles supports cautious extrapolation across these species in preclinical studies and may inform the design of future clinical investigations.

Values f_abs of ss-PHMGd following OI, estimated in guinea pigs and Chinchilla rabbits, was 5.9% and 8.9%, respectively. Comparison of PK parameters between guinea pigs and Chinchilla rabbits revealed no species specificity in the ADME of ss-PHMGd, as indicated by a bioavailability coefficient f_abs (ratio of AUC∞ values for extravascular and intravascular administration of a drug) close to 1 and equal to 0,7. This suggests the possibility of extrapolating data between these species in preclinical studies and potentially to humans, facilitating the interpretation of results and planning of subsequent possible clinical trials.

Dose proportionality analysis results

Data on the system parameter AUC∞, calculated by the software, for three doses of ³H-ss-PHMGd during OI into outbred guinea pigs are presented in Table 3.

Table 3.

Software calculated AUC∞ data for three doses of ³H-ss-PHMGd.

Pharmacokinetic parameter ± SD1	Dose, mg/kg (#TD2)
	0.0044 (0.5TD)	0.0089 (TD)	0.044 (5TD)
AUC∞3, min×µg/ml	4.7 ± 0.59	10.8 ± 1.45	57.0 ± 7.17
t1/24, min	1681.59 ± 233.57	1732.62 ± 254.65	1682.70 ± 233.67
CL5, ml/min/kg	0.989 ± 0.125	0.864 ± 0.116	0.819 ± 0.103
Cmax6, ug/ml	0.001881 ± 0.000068	0.004213 ± 0.000156	0.022735 ± 0.000821
Vd7, ml	2399.0 ± 449.9	2159.3 ± 430.0	1988.4 ± 373.6

¹standard deviation;

²therapeutic dose;

³area under the concentration vs time curve;

⁴elimination half-life;

⁵total clearance;

⁶maximum concentration;

⁷apparent volume of distribution.

The results of the weighted regression analysis yielded the following values: bw=1340.3 and aw=-1.13 with variances: D(bw)= 717.45 and D(aw)= 0.027. The corresponding t-values for hypothesis testing were: tobserved (bw)=50.0 and tobserved (aw)=−6.8, which were then compared with the critical table value t (a=0.05)=12.7.

The slope coefficient (bw) was statistically significant, and the intercept (aw) was not significantly different from zero. This supports the hypothesis that AUC∞ increases proportionally with dose in the tested range (0.5 to 5 TD).

The dose-dependent PK parameters AUC∞ and C_max increased with dose in a consistent and proportional manner, supporting a dose-proportional exposure profile. For example, a 10-fold increase in dose (from 0.0044 to 0.044 mg/kg) resulted in approximately a 12-fold increase in AUC (from 4.7 to 57.0 min × μg/ml) and a 12-fold increase in C_max (from 0.00188 to 0.02273 μg/ml). In parallel, the dose-independent parameters remained relatively invariant across the dosing range: 1) The elimination t₁/₂ remained stable across doses (range: 1681.6–1732.6 min); 2) The apparent clearance normalized to body weight (CL, ml/min/kg) showed only minor variability; 3) The V_d demonstrated a decreasing trend but remained within the expected range with overlapping standard deviations. These findings indicate no major deviation from linear PK over the investigated dose range. Thus, despite inherent biological variability, the systemically observed exposure is consistent with a dose-proportional kinetic profile.

The relationship between dose and systemic exposure was analyzed using the power model. β was 1.076, with a 90% confidence interval of 0.870 to 1.283. As the 90% CI includes 1, dose proportionality of AUC∞ can be concluded over the studied dose range (0.5TD to 5TD). The log-log plot of AUC∞ versus dose with the fitted regression line is shown in Figure 3.

Figure 3.

Log-log plot of AUC∞ versus dose for ³H-ss-PHMGd.

The power model analysis demonstrated that systemic exposure to ³H-ss-PHMGd increased proportionally with the administered dose within the studied range. β of 1.076 with the corresponding 90% CI (0.870–1.283) supports the linear PK of the compound. These results indicate that systemic exposure increases predictably with dose escalation, suggesting linear PK.

Tissue disposition studies

The q_critical for the three compared parameters (AUC∞, C_min, and C_max) are presented in Table 4.

Table 4.

The qqritical for the Newman-Keuls test, depending on the paired comparison variant in the ranked 1-2-3 segment of the uplink from the compared parameters..

PK parameter	qcritical (group comparison)
	q (1-3)	q (2-3)	q (1-2)
AUC∞1, min×µg/ml	3.34	2.79	2.79
Cmax2, µg/g	2.79	2.79	2.79
Cmin3, µg/g	3.77	3.08	3.08

¹area under the concentration vs time curve;

²maximum concentration;

³minimal concentration.

These values were compared with data obtained from the experiments (Table 5), where ³H-ss-PHMGd accumulated in kidneys, liver, thymus, heart, spleen, brain and lungs.

Table 5.

Values of compared PK parameters after the 1st, 2nd and 3rd administrations.

Organ	PK parameter	Value ± SD1			Group Comparison	q	qcritical	p	Significance
		1st adm2	2nd adm	3rd adm
Blood	AUC∞4	10.8 ± 1.45	10.4 ± 1.66	11.5 ± 1.68	1st vs 3rd	0.66	3.34	>0.05	NS3
					2nd vs 3rd	0.41	2.79	>0.05	NS
					1st vs 2nd	0.25	2.79	>0.05	NS
	Cmax5	0.0042 ± 0.0002	0.0043 ± 0.0002	0.0044 ± 0.0002	1st vs 3rd	1.16	3.34	>0.2	NS
					2nd vs 3rd	0.58	2.79	>0.5	NS
					1st vs 2nd	0.58	2.79	>0.5	NS
	Cmin6	0.00026±0.00003	0.00027±0.00001	0.00027±0.00005	1st vs 3rd	0.97	3.77	>0.2	NS
					2nd vs 3rd	0.23	3.08	>0.8	NS
					1st vs 2nd	0.74	3.08	>0.4	NS
Kidney	AUC∞	33.0±5.59	31.9±3.77	40.4±5.03	1st vs 3rd	1.75	3.34	>0.05	NS
					2nd vs 3rd	1.53	2.79	>0.05	NS
					1st vs 2nd	1.53	2.79	>0.05	NS
	Cmax	0.0133±0.0005	0.0139±0.0004	0.016±0.0005	1st vs 3rd	5.72	3.34	<0.01	**
					2nd vs 3rd	4.45	2.79	<0.05	*
					1st vs 2nd	1.27	2.79	>0.2	NS
	Cmin	0.00074±0.00009	0.00078±0.00006	0.00097±0.00005	1st vs 3rd	8.30	3.77	<0.001	***
					2nd vs 3rd	1.61	3.08	>0.1	NS
					1st vs 2nd	1.61	3.08	>0.1	NS
Liver	AUC∞	15.2±2.23	18.8±2.85	19.1±3.05	1st vs 3rd	1.46	3.34	>0.05	NS
					2nd vs 3rd	0.11	2.79	>0.05	NS
					1st vs 2nd	1.35	2.79	>0.05	NS
	Cmax	0.0067±0.0002	0.0078±0.0003	0.0085±0.0003	1st vs 3rd	6.45	3.34	<0.01	**
					2nd vs 3rd	2.61	2.79	<0.05	*
					1st vs 2nd	3.83	2.79	<0.05	*
	Cmin	0.00034±0.00004	0.00026±0.00006	0.00031±0.00004	1st vs 3rd	4.12	3.77	<0.001	***
					2nd vs 3rd	1.68	3.08	>0.1	NS
					1st vs 2nd	2.43	3.08	>0.05	NS
Skeletal muscles	AUC∞	12.7±1.93	0.0061±0.0002	0.0062±0.0003	1st vs 3rd	2.98	3.34	>0.05	NS
					2nd vs 3rd	1.55	2.79	>0.05	NS
					1st vs 2nd	1.43	2.79	>0.05	NS
	Cmax	0.0046±0.0002	0.00041±0.00004	0.00049±0.0001	1st vs 3rd	7.26	3.34	<0.01	**
					2nd vs 3rd	0.45	2.79	>0.05	NS
					1st vs 2nd	6.80	2.79	<0.05	**
	Cmin	0.0004±0.0001	17.4±2.26	21.0±2.73	1st vs 2nd	3.35	3.77	>0.05	NS
					1st vs 3rd	3.35	3.08	>0.05	NS
					2nd vs 3rd	0.24	3.08	>0.05	NS
Thymus	AUC∞	13.3±1.69	17.4±2.26	21.0±2.73	1st vs 2nd	3.39	3.34	<0.05	*
					1st vs 3rd	1.57	2.79	>0.05	NS
					2nd vs 3rd	1.81	2.79	>0.05	NS
	Cmax	0.004±0.0002	0.0055±0.0002	0.0067±0.0002	1st vs 2nd	13.47	3.34	<0.01	**
					1st vs 3rd	5.89	2.79	<0.01	**
					2nd vs 3rd	7.58	2.79	<0.01	**
	Cmin	0.0004±0.00005	0.0006±0.0001	0.0007±0.00012	1st vs 2nd	8.41	3.77	<0.01	**
					1st vs 3rd	3.41	3.08	<0.05	*
					2nd vs 3rd	5.00	3.08	<0.01	**
Heart	AUC∞	13.3±1.69	17.4±2.26	21.0±2.73	1st vs 2nd	3.39	3.34	<0.05	*
					1st vs 3rd	1.57	2.79	>0.05	NS
					2nd vs 3rd	1.81	2.79	>0.05	NS
	Cmax	0.004±0.0002	0.0055±0.0002	0.0067±0.0002	1st vs 2nd	13.47	3.34	<0.01	**
					1st vs 3rd	5.89	2.79	<0.05	*
					2nd vs 3rd	7.58	2.79	<0.05	*
	Cmin	0.0004±0.00005	0.0006±0.0001	0.0007±0.00012	1st vs 2nd	8.41	3.77	<0.001	***
					1st vs 3rd	3.41	3.08	<0.05	*
					2nd vs 3rd	5.00	3.08	<0.05	*
Spleen	AUC∞	12.6±1.93	11.4±1.93	15.7±2.17	1st vs 2nd	2.13	3.34	>0.05	NS
					1st vs 3rd	1.56	2.79	>0.05	NS
					2nd vs 3rd	0.57	2.79	>0.05	NS
	Cmax	0.0046±0.0002	0.0045±0.0002	0.006±0.0002	1st vs 2nd	7.09	3.34	<0.001	***
					1st vs 3rd	6.62	2.79	<0.001	***
					2nd vs 3rd	0.47	2.79	>0.05	NS
	Cmin	0.0004±0.0001	0.00027±0.00004	0.00034±0.00007	1st vs 2nd	6.15	3.77	<0.001	***
					1st vs 3rd	2.69	3.08	>0.05	NS
					2nd vs 3rd	3.46	3.08	<0.05	*
Brain	AUC∞	65.4±7.59	73.3±9.47	79.5±10.76	1st vs 2nd	1.50	3.34	>0.05	NS
					1st vs 3rd	0.66	2.79	>0.05	NS
					2nd vs 3rd	0.84	2.79	>0.05	NS
	Cmax	0.02±0.0008	0.027±0.0011	0.03±0.0012	1st vs 2nd	9.64	3.34	<0.001	***
					1st vs 3rd	2.89	2.79	<0.05	*
					2nd vs 3rd	6.75	2.79	<0.001	***
	Cmin	0.0004±0.0001	0.0017±0.0004	0.0024±0.0005	1st vs 2nd	13.91	3.77	<0.001	***
					1st vs 3rd	4.96	3.08	<0.01	**
					2nd vs 3rd	8.95	3.08	<0.001	***
Lungs	AUC∞	12.8±1.62	17.7±2.28	20.6±2.61	1st vs 2nd	3.54	3.34	<0.05	*
					1st vs 3rd	1.31	2.79	>0.05	NS
					2nd vs 3rd	2.23	2.79	>0.05	NS
	Cmax	0.004±0.0001	0.0053±0.0002	0.0065±0.0002	1st vs 2nd	15.87	3.34	<0.001	***
					1st vs 3rd	7.74	2.79	<0.001	***
					2nd vs 3rd	8.13	2.79	<0.001	***
	Cmin	0.0004±0.00004	0.0006±0.00009	0.0007±0.00012	1st vs 2nd	3.97	3.77	<0.05	*
					1st vs 3rd	2.59	3.08	>0.05	NS
					2nd vs 3rd	6.57	3.08	<0.001	***

¹standard deviation;

²administration;

³not significant;

⁴area under the concentration vs time curve;

⁵maximum concentration;

⁶minimal concentration;

*, ** or *** — if p < 0.05 / 0.01 / 0.001 respectively.

The calculated values of «q» that exceed the corresponding q_critical values from Table 4 are shown in bold. It should be noted that for heart, skeletal muscles, lungs, thymus and brain, an increase in the number of administrations led to an increase in AUC∞, C_max, C_min. In other cases this dependence was not so well traced.

The study found that ss-PHMGd was uniformly distributed across all major organs and tissues, with peak concentrations showing a maximum C_max ratio of 6.1 (brain tissue vs. thymus tissue). The highest concentrations were observed in brain and kidney tissues, while the lowest were in the thymus.

The results demonstrated higher levels of ss-PHMGd accumulation in the heart, brain, lungs, and thymus of outbred guinea pigs after OI, indicating a greater degree of penetration into these organs compared to others. Statistically significant increases in AUC∞, C_max, and C_min values were observed in the tissues of these organs following three daily instillations of the experimental drug. This may play a significant role in the context of potential systemic effects of the drug, especially with long-term use, which highlights the need to assess long-term safety with repeated administration.

Renal excretion study

The data of the studied parameters are presented in Table 6.

Table 6.

The main parameters of ³H-ss-PHMGd renal excretion after a single OI of TD in dosage form.

Excretion parameters	Animal number					Average value ± SD1
	1	2	3	4	5
Value of excretion, µg	0.19	0.17	0.19	0.17	0.22	0.19 ± 0.021
Value of excretion, %	5.4	4.9	5.4	4.8	6.3	5.3 ± 0.594
t1/2, h	29.1	25.9	36.3	27.4	25.4	28.8 ± 4.424
Maximal speed of excretion, µg/h	0.007	0.007	0.009	0.008	0.01	0.008 ± 0.001
Common diuresis, ml	153.9	151.9	161.8	171.4	171.3	162.1 ± 9.253

¹standard deviation;

²elimination half-life.

The average renal excretion of ss-PHMGd in experimental animals was 5.3%, indicating that approximately 95% of the administered dose is not accounted for through urinary elimination. This observation suggests that renal clearance is not the primary route of elimination for ss-PHMGd and points to the possibility of alternative excretory pathways or extensive distribution and retention in tissues. Given the cationic and polymeric nature of polyguanidine derivatives, it is likely that a significant portion of the compound binds to cellular and extracellular components, contributing to tissue retention. Another possible route is fecal excretion via the hepatobiliary system, which has been described for other highly charged or large molecular weight compounds with limited renal clearance. One report states that three male and female Rat/Alpk:APfSD (Wistar derived) animals with cannulated bile ducts received a single oral dose of unfractionated PHMB at 20 mg/kg body weight. The majority of radioactivity was excreted in feces (96.8% in males and 98.9% in females). Urinary excretion accounted for less than 3% of the administered radioactivity, and biliary excretion accounted for less than 0.2%. In another reported experiment, five males and five females received the low-molecular-weight fraction of PHMB, from which 7.8% of the administered dose was excreted in urine and 94.1% in feces in males, and 2.6% in urine and 93.5% in feces in females. In a different study conducted on male Alderley Park rats, the pattern of radioactivity excretion over 240 hours following a single oral dose of 20 mg/kg body weight/day of radiolabeled PHMB in five animals was as follows: 5.6 ± 0.35% was excreted in urine, 93.1 ± 1.58% in feces, and 0.2% via exhalation. Although the route of compound administration differed, these data appear to correlate with the findings of the present study, considering the similarity in the percentage of renal excretion and the comparable nature of the compounds [56]. It is important to note that in one study in rats, the majority of [14C]PHMB administered via drinking water or in the diet was absorbed; most of it was excreted in urine, with smaller amounts excreted in feces [55].

Conclusions

To the authors' knowledge, this is the first study to thoroughly investigate the systemic effects and PK profile of PHMG derivative in an ophthalmic drug formulation. The study examined the accumulation of the active component in various organs and tissues following OI of the experimental formulation, demonstrated a linear relationship of AUC-D parameters for three doses, and explored renal excretion. Given the proven efficacy of ss-PHMGd against eye infection pathogens, this research represents a critical step in assessing its systemic effects, which is essential for human toxicology studies and risk assessment, especially considering the negative history of polyguanidines in air humidifiers. Although the pharmacokinetic profile in the eye still needs further investigation, the tissue distribution of ss-PHMGd in other organs, studied in guinea pigs and chinchilla rabbits, shows promising potential for ophthalmic applications. The study demonstrated minimal penetration of the active compound into organs following ocular instillation. The lack of species-specific differences in ADME parameters between guinea pigs and chinchilla rabbits supports the broader applicability of these results and suggests consistent PK behavior across species. Additionally, the linearity of the AUC-D parameters simplifies dose optimization, which is crucial for clinical translation. A limitation of the present study is the absence of detailed physicochemical characterization of ss-PHMGd, including size distribution and polymer chain heterogeneity. Such profiling could further elucidate the relationship between molecular properties and pharmacokinetic behavior, particularly tissue penetration, and should be addressed in future studies. Overall, these findings lay a solid foundation for further clinical investigation of polyguanidines, highlighting their potential for ophthalmic treatments.