COLLAGEN HYDROLYSATE FOR USE IN PREVENTION AND/OR TREATMENT OF POST INTENSIVE CARE SYNDROME (PICS)

Abstract

The present invention relates to a use of collagen hydrolysate after intensive care unit (ICU) stay. The collagen hydrolysate improves the recovery and prevents and/or treats post intensive care syndrome (PICS) after ICU stay. The collagen hydrolysate is preferably administered as a supplement in the context of ICU stay and/or PICS. Collagen hydrolysate is generally-accepted to be safe and therefore can be taken as a preventive and long-term measure.

Claims

1. Collagen hydrolysate for use in prevention and/or treatment of post-intensive care syndrome (PICS) after intensive care unit stay.

2. Collagen hydrolysate for use according to claim 1, wherein the collagen hydrolysate is administered at 10-100 g total dose per day, based on the dry weight amount of the collagen hydrolysate.

3. Collagen hydrolysate for use according to claim 2, wherein the dosage regimen for collagen hydrolysate comprises administering collagen hydrolysate for at least 3 consecutive weeks after intensive care unit stay.

4. Collagen hydrolysate for use according to claim 2, wherein the dosage regimen for collagen hydrolysate comprises administration of collagen hydrolysate starting within 3 weeks after intensive care unit stay.

5. Collagen hydrolysate for use according to claim 2, wherein the dosage regimen for collagen hydrolysate comprises administering collagen hydrolysate at least once every day or at least once every other day.

6. Collagen hydrolysate for use according to claim 1, wherein the collagen hydrolysate has an average molecular weight of 1000-9000 Da.

7. Collagen hydrolysate for use according to claim 1, wherein the collagen hydrolysate has an average molecular weight of less than 5000 Da.

8. Collagen hydrolysate for use according to claim 7, wherein the collagen hydrolysate has an average molecular weight of 1400-2200 Da, preferably 1600-2000 Da.

9. Collagen hydrolysate for use according to claim 1, wherein the collagen is porcine collagen and/or the collagen hydrolysate is derived from hydrolysis of porcine collagen.

10. (canceled)

11. Use of collagen hydrolysate for improving the recovery after intensive care unit stay.

12. Use according to claim 11, wherein the collagen hydrolysate is administered at 10-100 g total dose per day, based on the dry weight amount of the collagen hydrolysate.

13. Use according to claim 12, wherein the dosage regimen for collagen hydrolysate comprises administering collagen hydrolysate for at least 3 consecutive weeks after intensive care unit stay.

14. Use according toclaim 12, wherein the dosage regimen for collagen hydrolysate comprises administration of collagen hydrolysate starting within 3 weeks after intensive care unit stay.

15. Use according to claim 12, wherein the dosage regimen for collagen hydrolysate comprises administering collagen hydrolysate at least once every day or at least once every other day.

16. Use according to claim 11, wherein the collagen hydrolysate has an average molecular weight of 1000-9000 Da.

17. Use according to claim 11, wherein the collagen hydrolysate has an average molecular weight of less than 5000 Da.

18. Use according to claim 17, wherein the collagen hydrolysate has an average molecular weight of 1400-2200 Da, preferably 1600-2000 Da.

19. Use according to claim 10, wherein the collagen is porcine collagen and/or the collagen hydrolysate is derived from hydrolysis of porcine collagen.

20-22. (canceled)

Description

FIGURE LEGENDS

[0122] FIG. 1. PICS score in the control, PICS, PICS+low dose CH and PICS+high dose CH groups (mean).

[0123] FIG. 2. Concentration-time curves of the free Hyp response [g/mL] (A) or total Hyp response [g/mL] (B) after intake of study products (mean, SD); n=6. CH=collagen hydrolysate; LMW=low molecular weight; HMW=high molecular weight.

[0124] FIG. 3. Human plasma levels of free glycine before and after oral ingestion of porcine-based hydrolysed collagen (1800 Da). Data are shown as mean+SEM (n=6).

EXAMPLES

Example 1: PICS Animal Study

Methods

Animal Model

[0125] A validated PICS mouse model was used, which is based on the model of Hujinami et al. (J Clin Med. 2021 Apr; 10(8): 1593.). The model is proven to induce PICS by mimicking ICU care following polymicrobial sepsis. This is achieved by intraperitoneal injection with cecal slurry followed by other typical ICU-related interventions (i.e. antibiotics and saline administration) for 72 h. The model incorporates a specific combination of physical (grip strength test), mental (anxiety level), and cognitive (discrimination index, habituation index, exploration time) parameters that often characterize PICS.

Pilot Study

[0126] The development of PICS in the model was first confirmed in a pilot study (not shown herein). The mice develop PICS after 1 week.

[0127] The molecular weight (i.e. 1800 Da) and source (porcine) of the collagen hydrolysate as used in this (full) comparative study is based on initial experiments comparing collagen hydrolysates of 1800 Da and 5000 Da and derived either from fish, bovine or porcine collagen sources. It was found that porcine collagen hydrolysate with relatively low molecular weight (<5000 Da, e.g. 1800 Da) has the largest putative effect in ameliorating PICS. Initial experiments showed that relatively early administration of collagen hydrolysate leads to most effective amelioration of PICS in the current model, as compared to delayed administration (delayed administration e.g. means starting from day 21 or later in the current model).

Treatment

[0128] The mice (C57BL6J, Charles River laboratories, France) receive collagen hydrolysate (CH, Peptan, Porcine 1800 Da, Rousselot B. V.) or the relevant controls according to the following experimental groups: [0129] 1. No PICS+vehicle (saline), i.e. Control [0130] 2. PICS+vehicle (saline), i.e. PICS [0131] 3. PICS+CH 1.8 g/kg (10 g clinical dose), i.e. PICS+low dose CH [0132] 4. PICS+CH 8 g/kg (44 g clinical dose), i.e. PICS+high dose CH The mice are administered once a day from day 12 through day 47.

[0133] The collagen hydrolysate used in Example 1 has the same composition as used in the clinical study in Example 3 (Table 2).

[0134] Treatment is started 4 days after the induction of PICS. The CH or vehicle is administered by daily oral gavage for a duration of 7 weeks (day 12-47).

[0135] In a follow-up study, the efficacy of group 3 (1.8 g/kg CH) is compared to that of fluoxetine (20 mg/kg) and whey protein (high in cysteine, 1.8 g/kg), administered by the same method.

Assessment of PICS

[0136] PICS sub-component #1 was determined with a grip strength meter (UGO Basile) to measure the forelimb grip strength, and the maximal force (g) was recorded. The mouse was pulled slightly backward by the tail while the forelimbs grasped the bar, which triggers a counter pull and automatically records the peak tension. Measurements were replicated in triplicate, and the maximum force of the three measurements were recorded. Data are quantified in % of grip loss.

[0137] PICS sub-component #2 relates to PICS-associated changes in the level of anxiety. Mice were subjected to an open-field test (Ethovison XT, Noldus) with an open field of 464640 cm divided into peripheral and central zones. The time spent in the central and peripheral zone is recorded. PICS sub-component #2 was calculated as the ratio of the time spent in the center over the total movement time and expressed as a percentage of the total movement time. Lower time spent in the central region is indicative of greater anxiety levels.

[0138] PICS sub-component #3, #4, #5 relate to PICS-associated behavioral changes. The object recognition test was conducted using an open field comprised of a box 464640 cm and a digital camera was used to record behavioral videos. Videos were recorded, and the time spent by mice with each object was calculated. First, the mice were allowed to explore and habituate to the empty open field box. Next, the test consists of two sessions (10 min each with 30 min intervals between each session). In the training session (acquisition phase), two similar objects were placed, and mice were allowed to explore them. In the test session (test phase), one object was replaced with a novel object. The interaction of the mouse with both objects (familiar and novel) was recorded. Exploratory activity and time spent sniffing each object during training and testing sessions were scored by Ethovision XT software. The percent discrimination index was calculated as 100 time spent exploring the novel object minus the time spent exploring the familiar object (TnovelTfamiliar) divided by total exploration time (Tnovel+Tfamiliar). Index of habituation indicates familiar object recognition and is calculated (based on A Ennaceur et al. Behav Brain Res. 1988 Nov. 1; 31(1):47-5) as the difference between average time spent exploring the objects in training and exploring novel objects in a test phase.

[0139] The PICS parameters were investigated over a period of 3-7 weeks after the induction of PICS. Table 1 shows the PICS score as normalized to the control group.

[0140] Results are shown for a sample size of 9-11.

Results

[0141] FIG. 1 shows the PICS score in the control, PICS, PICS+low dose CH and PICS+high dose CH groups. It can be seen that overall PICS is reduced by 38% in the PICS+low dose CH group and by 23% in the PICS+high dose CH group, compared to the PICS group (i.e. without collagen hydrolysate). Thus, both CH doses are effective in ameliorating PICS.

[0142] Table 1 shows the improvement in individual PICS parameters in the PICS+low dose CH and PICS+high dose CH groups, compared to the PICS group. It can be seen that both doses overall improve the individual PICS sub-components.

TABLE-US-00001 TABLE 1 Improvement (%) in individual PICS parameters for the low dose and high dose CH groups, compared to the PICS group (without CH administration) PICS sub-component Low dose CH High dose CH #1 15% 15% #2 43% 14% #3 78% 84% #4 47% 27% #5 19% 25% average 40% 28%

[0143] The effects of high dose CH are compared to fluoxetine and whey proteins, i.e. agents with demonstrated efficacy in conditions related to PICS. Fluoxetine has been shown to offer neuroprotection in conditions of stress, emotion, and affective behaviours in mice with PICS-related symptoms (Wang et al. Aging (Albany NY). 2021 Feb. 22; 13(6):8720-87360). It is therefore hypothesized that fluoxetine can ameliorate PICS by restoring mental and cognitive health in the PICS model. Whey protein has been suggested to promote healing and recovery in critically ill patients, e.g. by limiting muscle atrophy and promoting metabolic stability (Tsutsumi et al. JPEN J Parenter Enteral Nutr. 2015 Jul;39(5):552-61). It is therefore hypothesized that fluoxetine can ameliorate PICS by restoring physical health in the PICS model.

[0144] As expected, in contrast to collagen hydrolysate, treatment with whey protein or fluoxetine leads to little/no improvement in PICS score. This shows that agents targeting only certain sub-components or individual symptoms of PICS cannot ameliorate PICS as a whole.

[0145] The PICS sub-component #1 was evaluated at week 1 and week 3-7 for all groups (6-time points). Mostly starting from week 5 an improvement in PICS sub-component #1 was seen. Similar timing effects were also seen for the other PICS sub-components.

Example 2: Effect of Collagen Hydrolysate Source and Molecular Weight

Objective

[0146] To investigate the single-dose bioavailability of collagen hydrolysate from different sources and of different mean molecular weight. It is considered that the ability of a collagen hydrolysate to ameliorate PICS is associated with this bioavailability.

Methods

[0147] Bioavailability was assessed by evaluating the uptake of free and peptide-bound hydroxyproline (Hyp) as marker amino acid. A randomized, double blind, cross-over clinical study was performed with healthy volunteers.

[0148] A single-dose of 10 g collagen hydrolysate was provided at low mean molecular weight (2000 Da). For the bovine collagen hydrolysate, a single-dose of 10 g was provided of either low mean molecular weight (2000 Da, LMW) or high mean molecular weight (5000 Da, HMW). Blood was sampled for analysis over a period of 6 hours after collagen hydrolysate ingestion.

Results

[0149] FIG. 2 shows the concentration-time curves of the free Hyp response (A) or total Hyp response (B) after intake of study products.

[0150] It was found that, by intake of 10 g of the collagen hydrolysate, free Hyp concentrations in plasma (C.sub.max) were greatly increased with an average factor of 7.2 for fish, 9.9 for porcine and 6.2 for both bovine low molecular weight (LMW) and high molecular weight (HMW) collagen hydrolysate. With respect to the bioavailability of free Hyp, there were no significant differences between the incremental area under the curve (iAUC) of the investigated products. In addition, Hyp content in blood samples was determined after total hydrolysis. Significantly higher concentrations of total Hyp were determined comparing the iAUC of free and total Hyp. The total Hyp concentrations in plasma (C.sub.max) were also greatly increased for either fish and porcine collagen hydrolysate, and both for bovine collagen hydrolysate LMW and bovine collagen hydrolysate HMW.

[0151] Overall, the results show that the uptake of collagen hydrolysates in blood appears similar for collagen hydrolysates from the sources porcine, bovine and fish and also for hydrolysates with a relatively high average molecular weight (5000 Da) and relatively lower average MW (2000 Da).

Example 3: PICS Clinical Study

Objective

[0152] Based on the findings of the PICS animal model, a clinical study is performed to verify the use of collagen hydrolysate in the prevention and/or treatment of PICS in a clinical setting. The aim is to study the effect of six weeks intervention of porcine collagen hydrolysate administration versus control (maltodextrin administration) on PICS after ICU stay.

Methods

Study Design

[0153] Randomized controlled (double-blind) trial in 72 ICU-patients >18 years old discharged from the ICU (minimum ICU stay of 72 h). One group receives twice daily 22 g porcine collagen hydrolysate (20 g proteins, 80 kcal), so in total 44 g (40 g proteins, 160 kcal) per day. The other group receives twice daily 21 g maltodextrin (0 g protein, 82 kcal), so in total 42 g (0 g protein, 164 kcal). Interventions are isocaloric. Collagen hydrolysate or maltodextrin is provided in the morning and the afternoon.

Investigational Product

[0154] The porcine collagen hydrolysate is provided in a powdered form. The powder contains approximately 97% protein of dry weight. The porcine protein supplement (per 100 g) contains, on average: 360 kcal, 0 g fats, 0 g carbs (of which 0 g sugar), 90 g proteins and 0 g fibers. The amino acid content can be found in Table 2 below. The collagen hydrolysate has an average molecular weight of 1800 Da.

[0155] The control group will receive maltodextrin daily for six weeks. Maltodextrin is a polysaccharide, and it is also provided as a white powder. The intervention product consists of primary proteins and no carbs, and maltodextrin contains carbs and no proteins. Maltodextrin (per 100 g) contains, on average: 390 kcal, 0 g fats, 97.5 g carbs (of which 8.8 g sugar), 0 g proteins and 0 g fibers. The collagen hydrolysate and maltodextrin have similar appearances and the doses provided in the study are isocaloric.

TABLE-US-00002 TABLE 2 Amino acid content of the collagen hydrolysate g/100 g g/22.22 g g/44.44 g Amino acid protein protein protein Alanine 9.1 2.02202 4.04404 Arginine 8 1.7776 3.5552 Aspartic acid 5.1 1.13322 2.26644 Glutamic acid 10.1 2.24422 4.48844 Glycine 22.3 4.95506 9.91012 Histidine 1.6 0.35552 0.71104 Hydroxylysine 0.8 0.17776 0.35552 Hydroxyproline 10.2 2.26644 4.53288 Isoleucine 1.3 0.28886 0.57772 Leucine 2.8 0.62216 1.24432 Lysine 3.7 0.82214 1.64428 Methionine 0.9 0.19998 0.39996 Phenylalanine 2 0.4444 0.8888 Proline 14.3 3.17746 6.35492 Serine 3.4 0.75548 1.51096 Threonine 1.9 0.42218 0.84436 Tyrosine 0.5 0.1111 0.2222 Valine 2.3 0.51106 1.02212

Route of Administration

[0156] The collagen hydrolysate or maltodextrin is provided in powder form packaged in identical sachets. Each administration sachet contains 22 g, each maltodextrin sachet contains 21 g. Both powders are freely soluble so that they can be dissolved in liquids. For example, 10 g collagen hydrolysate can easily be dissolved in 200 ml of lemonade with just a little stirring, and even higher amounts are possible (more concentrated).

Assessment of PICS

[0157] PICS is assessed according to a measuring instrument of the local hospital, which uses a combination of the following parameters: [0158] composite score consisting of handgrip strength (Jamar dynamometer); [0159] muscle strength leg (HHD m. quadriceps fem); [0160] muscle strength arm (HHD m. biceps brachii); [0161] exercise capacity (6MWD); [0162] handgrip strength (Jamar dynamometer); [0163] muscle strength leg (HHD m. quadriceps fem); [0164] muscle strength arm (HHD m. biceps brachii); [0165] exercise capacity (6MWD); [0166] lower extremity muscle strength (TCST); [0167] CPAx; [0168] MRCsum; [0169] Health related quality of life (assessed by EQ-5D); [0170] Barthel score; [0171] Rockwood Clinical Frailty Scale.

Bioabsorption of Amino Acids

[0172] The time-course appearance of free glycine in the human plasma after the ingestion of the collagen hydrolysate was measured in six subjects hourly.

Outcome

[0173] The clinical study uses the same administration scheme as the PICS animal model in Example 1. The administration of collagen hydrolysate after ICU stay improves recovery from critical illness and prevents PICS from developing. From earlier clinical studies, the administration of collagen hydrolysate and the dose is considered safe (e.g. based on adverse events, lab creatinine, urea, CK, glucose, Hb, CRP). The clinical study confirms earlier results and collagen hydrolysate can be specifically used in ICU survivors to prevent and/or treat PICS in daily practice.

[0174] After ingestion of collagen hydrolysate, it was found that glycine levels increased to the maximum concentration at one hour time point. Subsequently, the glycine declines close to the base level over the course of several hours (FIG. 3). This observation suggests that one of the significant (abundant) amino acids absorbs quickly and is absorbed in and/or eliminated from the body over time.