FormalPara Key Summary Points

Lutein (L) and zeaxanthin (Z), known as macular carotenoids, are found in the macula of the human eye and are known to improve visual functions.

Poor bioavailability of supplemental L and Z poses a challenge to achieve significant benefits after consumption.

The study was conducted to evaluate the bioavailability of L and Z from a novel formulation known as Ocusorb® in comparison to the market sample.

Ocusorb® demonstrated superior bioavailability for lutein—2.5 times higher Cmax, 2.9 times higher AUC0–72, and 3.2 times higher AUC0–t compared to the market sample.

Ocusorb® demonstrated superior bioavailability for zeaxanthin—1.8 times higher Cmax, 2.2 times higher AUC0–72, and AUC0–t compared to the market sample.

Enhanced bioavailability from Ocusorb® can be beneficial for individuals looking to quickly improve their L and Z status and enhance their vision performance.

Introduction

Lutein and zeaxanthin (L and Z) are nutritionally important carotenoids found in several fruits and vegetables. As a result of selective accumulation in the macula of the human retina, L and Z are also known as macular carotenoids [1, 2]. L and Z have antioxidant properties and are known to absorb high energy blue light and protect the retina in the eye. The benefits of L and Z have been extensively researched for their roles in ocular, cognitive, and skin health [1, 3,4,5]. L and Z accumulate in the macula of the human eye and enhance visual performance [3, 5,6,7,8,9,10,11]. L and Z have also been shown to reduce the risk of age-related macular degeneration (AMD) [6, 7]. Choroidal neovascularization (CNV) plays a critical role in the pathogenesis of AMD, and in vivo animal models for neovascular AMD demonstrated significant suppression of the inflammatory process associated with CNV in response to lutein supplementation [12]. Regular intake of carotenoid-rich food has been associated with the reduced risk of several chronic diseases like photosensitivity diseases, cataracts, AMD, and cardiovascular diseases [13, 14].

L and Z are isomers with identical chemical formulas. Lutein occurs as a single stereoisomer, while zeaxanthin occurs as a mixture of three stereoisomers, namely (3R,3′R)-zeaxanthin, (3R,3′S)-zeaxanthin, and (3S,3′S)-zeaxanthin. L and Z from the diet reach plasma and subsequently the macula where lutein is also converted to meso-zeaxanthin. Thus, the macular carotenoids responsible for macular pigment optical density (MPOD) are lutein, zeaxanthin, and meso-zeaxanthin at an approximate ratio of 2:1:1 in the retina [15]. Humans cannot synthesize lutein and (3R,3′R)-zeaxanthin and are dependent on dietary sources such as spinach, corn, lettuce, green peas, kale, pepper, orange, zucchini, and egg yolk to support adequate L and Z status [16, 17]. However, the intake of these fruits and vegetables are below the recommendations and dietary L and Z consumption is very low in most parts of the world. Inadequate dietary intakes of L and Z resulting in low serum levels of L and Z are associated with low macular pigment density and increased risk of AMD [18]. Supplementation with L and Z can help to support adequate L and Z status [19,20,21] and have been clinically demonstrated through increased serum levels of L and Z as well as higher deposition in the retina [3, 10]. However, considerable inter-individual variation in serum concentrations and macular pigment density has been reported following supplementation with L and Z [10]. Factors that affect the absorption of carotenoids, such as the matrix of the formulation and the form (i.e., free versus esterified) of the carotenoids determine the amount of L and Z achieved in serum after supplementation [4, 11]. Moreover, L and Z have poor bioavailability due to poor stability and solubility of the crystalline form in an aqueous medium. Hence, there has been considerable effort to develop L and Z formulations with improved stability and enhanced bioavailability to achieve improved health benefits. Various methods that have been used to develop formulations for L and Z include dispersion of crystals in edible oils, encapsulation with biopolymers [22], or mechanochemical complexation [23,24,25,26].

Here we report a novel formulation of L and Z (LZO) with improved bioavailability (patent filed WO 2021/074763 A1) using an optimized marigold and paprika extract as a source of L and Z respectively at a 5:1 ratio as found in nature in the form of natural dietary sources [27]. We used a micronization process to reduce the particle size followed by formation of an oil suspension in medium chain triglyceride (MCT) oil along with a combination of functional oils that helps to improve absorption from the small intestine. We evaluated the bioavailability of LZO in healthy human volunteers after single oral dose as compared to commercially available formulation (LZC) with L and Z at a similar dose.

Methods

The LZO formulation (Ocusorb®) was made using excipients like sunflower lecithin, olive oil, mixed tocopherol, thyme oil, linseed oil, and lime peel extract oil dissolved in MCT oil followed by addition of micronized marigold/paprika oleoresin mixture. This finished formulation was used for preparation of the study capsules. The final preparation was analyzed by HPLC using reference standard to ensure each gram of final product contains 80% of lutein and 16% of zeaxanthin. Each capsule (LZO) contained 10 mg of lutein and 2 mg of 3′R,3R-zeaxanthin. The reference group utilized LZC capsules which contained a blend of commercially available sample of lutein 20% and zeaxanthin 14% to obtain the dose of 10 mg lutein and 2 mg RR-zeaxanthin per capsule.

Study Design and Procedures

This was a double-blind, balanced, randomized, two-treatment, single-period, single-dose, parallel, comparative bioavailability study in healthy, adult, human subjects under fed conditions. The study was initiated after obtaining written approval from an institutional ethics committee, Maarg Independent Ethics Committee (EC registration Number ECR/77/Indt/AP/2013/RR-19), Secunderabad, India on 18 November 2019. The study was carried out in compliance with the requirements of the Indian Council of Medical Research (ICMR) ethical guidelines, International Council for Harmonization (ICH) Guidance on Good Clinical Practice (E6R2), and Declaration of Helsinki. The study was registered with the Clinical Trials Registry of India (CTRI/2019/11/022082).

Informed consent was voluntarily obtained from every participant before enrolling for the study. Subjects were randomly assigned in a 1:1 ratio to receive either LZO or LZC. The randomization schedule was generated by a non-study assigned, independent expert ensuring the treatment balance using SAS® statistical software, version 9.4. Staff who were involved in the investigational product-related activities were not involved in the study-related activities to ensure double blindness of the study.

Subjects stayed at the study center for a total of 6 days, 3 days before dosing and 3 days after dosing. During their stay, subjects were provided standard meals to control lutein and zeaxanthin in the diet. Subjects fasted overnight for at least 10 h and then received a high-fat, high-calorie breakfast comprising of 900 kcal with 57% fats, 27% carbohydrates, and 16% proteins the next day morning and 30 min later were administered a single oral dose of LZO or LZC containing 10 mg L or 2 mg Z along with 240 ± 2 mL of water. The subjects were dosed in a parallel manner to either LZO or LZC in the single period. The study product was administered to the subjects in a sitting posture and they remained in a sitting posture for the first 2 h (except at the time of vitals, natural and medical emergencies) unless clinically indicated. Thereafter, the subjects were allowed to engage only in normal activities while avoiding severe physical exertion. Blood test, vitals, and medical examinations were done for safety analysis.

Fourteen blood samples of 1 × 6 mL were collected from each subject. Three pre-dose blood samples were collected at 48, 24, and 0 h before dosing to establish baseline levels of lutein and zeaxanthin. Post-dose blood samples were collected at 2, 4, 6, 8, 10, 12, 16, 20, 24, 48, and 72 h. The blood sample collection and analysis were carried out under yellow monochromatic light because of the light sensitivity of lutein and zeaxanthin. After sample collection, samples were placed at room temperature for up to 30 min (time required for proper clot formation) and then centrifuged at 4 °C and 4000 rpm for 10 min to separate serum. The samples were placed in a deep freezer maintained at − 70 °C ± 10 °C until completion of serum sample analysis. A bioanalytical method for the estimation of lutein and zeaxanthin in human serum was developed and validated as per regulatory guidelines. Lutein and zeaxanthin were extracted from human serum by a liquid–liquid extraction method. Astaxanthin was used as internal standard for both lutein and zeaxanthin. The separation was achieved using a Kromasil 100-5SIL 250 × 4.6 mm, 5 μm with hexane/ethyl acetate (55:45 v/v) as mobile phase. The measured concentrations for each subject for all the time points were calculated against the calibration curve prepared with known standards. The retention time was 5.85, 6.48, and 3.90 min for lutein, zeaxanthin, and astaxanthin, respectively. Lutein and zeaxanthin were detected at wavelength of 456 nm and their concentrations in human serum samples were quantified using LabSolution 6.86 SP2 with a Shimadzu HPLC–photodiode array detection (PDA) system. The method was linear and validated over a concentration range of 20.92 to 10,434.15 ng/mL for lutein and 20.05 to 9998.85 ng/mL for zeaxanthin.

Study Population and Inclusion/Exclusion Criteria

A total of 90 healthy adult human subjects were enrolled in the study in accordance with the inclusion and exclusion criteria listed in Table 1.

Table 1 Inclusion and exclusion criteria for subject selection
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Statistical Analysis

A sample size of 41 subjects per arm (82 subjects for both arms) was sufficient considering the difference in means and standard deviation with a 0.05 as alpha, two-sided significance level, and 90% as power. However, considering the 10% dropouts due to non-compliance or due to personal reasons, 90 subjects were randomized and dosed.

Study conduct and statistical analysis were performed at AnaCipher Clinical Research Organisation, Hyderabad using the SAS system for windows, version 9.4 (SAS institute Inc., Cary NC, USA). Pharmacokinetics parameters (Cmax, AUC0–72, AUC0–t, Tmax, and t1/2) were determined from the serum lutein and zeaxanthin concentrations by using Phoenix WinNonlin Version 8.2.

The arithmetic mean, minimum, maximum, standard deviations, and geometric mean were reported for the pharmacokinetic parameters of untransformed data. The geometric means were reported for AUC0–72, AUC0–t, and Cmax of log-transformed data.

The log-transformed pharmacokinetic parameters (AUC0–72, AUC0–t, and Cmax) were analyzed using an analysis of variance (ANOVA) model with the main effects of treatment. A 5% level of significance was used throughout the analysis. Each analysis of variance included calculation of least-square means, adjusted differences between treatment means, and the standard error associated with these differences.

For the pharmacokinetic parameters (AUC0–72, AUC0–t, and Cmax), 90% confidence intervals for the ratios of LZO and LZC averages were calculated using the ANOVA output from the analysis of the log-transformed data.

The 90% confidence intervals for the ratio of geometric least squares means between the formulations were calculated, for log-transformed pharmacokinetic parameters—Cmax, AUC0–72, AUC0–t for lutein and zeaxanthin.

The p values were calculated using the PROC GLM (ANOVA) output from the analysis of the log-transformed data. Probability values less than 0.05 denote statistically significant differences between LZO and LZC.

Safety assessments included monitoring of adverse events, physical examination, vital signs measurements, and laboratory assessments.

Results

Ninety male subjects completed the study with 100% compliance (Fig. 1). Out of the 90 randomized subjects, 45 subjects were allocated to the LZO group and 45 subjects to the LZC group. The demographic characteristics are provided in Table 2. The mean age of subjects was 29.96 ± 6.28 years in the LZO group and 31.00 ± 5.41 years in the LZC group. The mean body mass index (BMI) was 23.76 ± 2.30 in the LZO group and 23.55 ± 2.08 in the LZC group. There was no statistically significant difference between the two groups for age, and BMI at the baseline. Female subjects did not participate in the study because of social reasons associated with long-term housing at the study center.

Fig. 1
figure 1

Flowchart of this randomized, double-blind, comparative bioavailability study of lutein and zeaxanthin formulations

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Table 2 Demographic characteristics of the subjects
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The analysis of pharmacokinetic parameters is presented for lutein and zeaxanthin in Table 3.

Table 3 Results of baseline-corrected pharmacokinetic parameters for lutein and zeaxanthin
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Results for Lutein

The LZO group showed significantly (p < 0.05) higher lutein serum concentrations (> 2-fold) at all time points i.e., 2 to 72 h post dose as compared to the LZC group (Fig. 2a).

Fig. 2
figure 2

Lutein and zeaxanthin serum levels versus time. The LZO group showed significantly (p < 0.05) higher serum lutein levels (> 2-fold) at all time points i.e., 2–72 h post dose (a) and higher serum zeaxanthin levels (> 1.5-fold) from 4 to 72 h post dose (b) as compared to the LZC group

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The maximum serum concentration (Cmax) was significantly (p < 0.05) higher for LZO as compared to LZC (219.15 vs. 87.01 ng/mL; 90% CI 191.97–330.44%). Similarly, the total systemic exposure measured as area under the curve (AUC) was significantly (p < 0.05) higher for LZO as compared to LZC represented by AUC0–72 (8499.89 vs. 2892.09 h·ng/mL; 90% CI 217.33–397.46%) and AUC0–t (8264.02 vs. 2622.97 h·ng/mL; 90% CI 224.50–442.17%), respectively.

The LZO group showed significantly (p < 0.05) higher absorption for lutein based on 2.5 times higher Cmax, 2.9 times higher AUC0–72, and 3.2 times higher AUC0–t levels compared to the LZC group. (Table 3, Fig. 3).

Fig. 3
figure 3

Pharmacokinetic parameters of lutein in serum for test product (LZO) and reference product (LZC). a Mean serum lutein maximum concentration (Cmax). b Lutein area under the curve (AUC) serum concentration versus time for 0–72 h (AUC0–72) and 0 to last measurable concentration (AUC0–t). Cmax, AUC0–72, and AUC0–t were significantly (p < 0.05) higher for LZO compared to LZC

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The median Tmax value for LZO was 20 h and LZC was 16 h for Lutein.

Results for Zeaxanthin

The LZO group showed significantly (p < 0.05) higher serum zeaxanthin concentrations (> 1.5-fold) from 4 to 72 h post dose as compared to the LZC group (Fig. 2b).

The maximum serum concentration (Cmax) was significantly (p < 0.05) higher for LZO as compared to LZC (56.95 vs. 32.55 ng/mL; 90% CI 140.70–217.50%), respectively. Similarly, the total systemic exposure measured as area under the curve (AUC) was significantly (p < 0.05) higher for LZO when compared to LZC represented by AUC0–72 (1929.93 vs. 861.65 h·ng/mL; 90% CI 159.64–314.26%) and AUC0–t (1817.29 vs. 814.17 h·ng/mL; 90% CI 154.35–322.79%), respectively.

The LZO group showed significantly (p < 0.05) higher absorption for zeaxanthin with 1.8 times higher Cmax, 2.2 times higher AUC0–72, and 2.2 times higher AUC0–t levels as compared to the LZC group (Table 3, Fig. 4).

Fig. 4
figure 4

Pharmacokinetic parameters of zeaxanthin in serum for test product (LZO) and reference product (LZC). a Mean serum zeaxanthin maximum concentration (Cmax). b Zeaxanthin area under the curve (AUC) serum concentration versus time for 0–72 h (AUC0–72) and 0 to last measurable concentration (AUC0–t). Cmax, AUC0–72, and AUC0–t were significantly (p < 0.05) higher for LZO compared to LZC

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The median Tmax for LZO was 20 h and LZC was 24 h for Zeaxanthin.

The study product was well tolerated and did not show any adverse events.

Discussion

L and Z, also known as macular carotenoids, support visual performance and provide protection from ocular diseases such as AMD and cataract [1, 2, 28, 29]. Multiple human pharmacokinetic studies of L and Z have shown that intake of L and Z through diet or supplementation increases circulating levels of L and Z in the body leading to enhanced deposition in the macula of the eye. However, L and Z in the natural form have low bioavailability after oral intake due to their poor stability and inefficient absorption in the intestine leading to varying plasma concentrations [30]. In the current study we report a highly bioavailable formulation of L and Z that demonstrated significant improvement in absorption leading to increased serum levels after a single oral dose.

We observed a significant (p < 0.05) increase in serum lutein (> 2-fold) and zeaxanthin (> 1.5-fold) concentrations in the case of the LZO formulation at all time points measured post dose as compared to the LZC group. The maximum serum lutein concentration (Cmax) for LZO was 219.15 ng/mL which was 2.5 times higher and statistically significant (p < 0.05) as compared to LZC (Cmax 87.01 ng/mL). Similarly, maximum serum zeaxanthin concentration was 1.8 times higher with a statistically significant (p < 0.05) increase (Cmax 56.95 ng/mL) in the case of LZO as compared to LZC (Cmax 32.55 ng/mL). Further area under the curve (AUC) measurements for both AUC0–72 and AUC0–t, which is the total analyte exposure across time, significantly increased absorption for both L and Z in the case of LZO as compared to the LZC formulation. The LZO group showed 2.9 times higher AUC0–72 and 3.2 times higher AUC0–t levels for lutein and 2.2 times higher AUC0–72 and AUC0–t levels for zeaxanthin as compared to the LZC group.

The time taken for lutein to reach its highest concentration in serum (Tmax) was 20 h in the case of LZO as compared to 16 h in the case of LZC. Similarly, Tmax for zeaxanthin was 20 h in the case of LZO and 24 h for LZC. Overall, we observed an increased serum L and Z levels in the case of the LZO formulation as a result of faster and better absorption after supplementation which may aid better accumulation in the macula and enhance eye protection. Increased serum L and Z levels are known to increase the MPOD and directly improve visual performance [31].

Several studies have reported variable bioavailability of carotenoids due to the type of formulation matrix used, form of the bioactives (free versus esterified), and effects of other nutrients [32]. We micronized marigold and paprika oleoresin (source of L and Z, respectively) to reduce the particle size which in turn is known to improve dissolution rate and enhance oral bioavailability [33, 34]. Lutein in free form is not very stable and hence we used an oil suspension of medium chain triglycerides (MCT). MCT oil is considered as a healthy fatty acid, known to enhance permeability of intestinal mucosal membrane [35], improve absorption of lutein [36], and widely used in the nutraceutical industry [35, 37]. Further, we also incorporated multiple functional oils in the formulation that are known to improve absorption in the gut [38,39,40]. We believe that the cumulative effect of reduced particle size and combination of excipients used in the formulation improved the bioavailability of L and Z in the LZO formulation.

Both free and esterified lutein have been used to study single-dose, pharmacokinetic (PK) properties in human subjects [41,42,43]. Although variables such as intestinal absorption, metabolism, and serum clearance of carotenoids have an impact on circulating serum levels, the dose of L and Z used is also directly correlated with serum levels achieved, with an increased dose resulting in higher serum concentration [41]. Further, at a higher dose, the free form of lutein is better absorbed than lutein esters leading to increased plasma lutein levels [27, 44]. In the current study we observed increased circulating levels of L and Z owing to our improved formulation which is significant as this is achieved without increasing the dose. Higher circulating levels of L and Z are known to increase MPOD levels leading to improved visual protection and performance. The National Eye Institute sponsored the AREDS2 clinical study to evaluate the protective effects of carotenoids on progression of eye diseases. The study reported that 5-year supplementation of 10 mg lutein and 2 mg zeaxanthin leads to 18% reduction in AMD progression [29]. Other studies reported that increased dose of L and Z supplementation leads to increased plasma circulating levels of L and Z and improved MPOD [32] with improved health benefits such as improved contrast sensitivity, disability glare performance, and photo stress recovery [45, 46].

A limitation of the study was that female subjects did not participate because of social reasons associated with long-term housing at the study center. However, gender-specific differences in blood concentrations of L and Z have been reported to be very minimal [47,48,49].

Conclusions

L and Z protects the retina from blue light and provides antioxidant and anti-inflammatory support to retina and choroidal blood vessels that supply blood to the macular region of the retina [12]. Increased intake of L and Z not only increases the MPOD in the macula [28, 32] leading to improved visual acuity, sensitivity, and glare [29, 30, 50,51,52] but also reduces the risk of AMD [28, 53,54,55,56,57,58,59]. We developed an improved formulation of L and Z with significantly improved oral absorption leading to higher blood levels of L and Z which may further help in increased MPOD levels. L and Z supplements are generally considered to be safe [32] and are well tolerated. The enhanced bioavailability of this proprietary formulation can be advantageous for individuals looking to quickly improve their L and Z status and enhance their vision performance. Further, long-term human studies that evaluate MPOD and other related eye health benefits should help in accurate prediction of long-term benefits of our improved formulation.