WO2023067349A1

WO2023067349A1 - Treatment of connective tissue injury

Info

Publication number: WO2023067349A1
Application number: PCT/GB2022/052683
Authority: WO
Inventors: Mohamed Hamza NOORDEEN
Original assignee: Noordeen Mohamed Hamza
Priority date: 2021-10-20
Filing date: 2022-10-20
Publication date: 2023-04-27
Also published as: GB202115026D0

Abstract

Methods of treatment of connective tissue injury in a subject are described, in particular, methods of treatment of tendon and ligament injury in humans and horses. The methods comprise administering to the subject an effective amount of vitamin A and platelet-rich plasma (PRP). Combined preparations and pharmaceutical compositions for use in the methods are also described.

Description

Treatment of Connective Tissue Injury

This invention relates to methods of treatment of connective tissue injury in a subject, in particular, treatment of tendon and ligament injury in humans and horses, and to combined preparations and pharmaceutical compositions for use in the methods.

Tissue repair encompasses two separate processes: regeneration and replacement. Regeneration refers to a type of healing in which new growth completely restores portions of damaged tissue to their normal state. Replacement refers to a type of healing in which severely damaged or non-regenerable tissues are repaired by the laying down of connective tissue, a process commonly referred to as scarring. Whilst some types of tissue injury (such as minor paper cuts) can sometimes be healed in such a way that no permanent damage remains, most tissue repair consists of both regeneration and replacement. Tissue repair may restore some of the original structures of the damaged tissue (such as epithelial layers), but may also result in structural abnormalities that impair function (such as the scar formed in the healing of a myocardial infarction).

Connective tissue injuries, such as tendon and ligament injuries, are some of the most common orthopaedic problems, and cause substantial pain, disability, and time off work. These type of injuries are also a particular problem in horses. Whilst many connective tissue injuries are acute, a large number are chronic, degenerative conditions. In either case, repair results in the formation of a fibrovascular scar that never attains the characteristics of normal tissue. Scarring is often an inevitable feature of wound healing, particularly if the wound is deep and/or infected, or chronic. Briefly, there are three main stages of wound healing: inflammation, new tissue formation, and remodelling. The inflammatory stage involves the formation of a blood clot, clearance of debris, and recruitment and activation of immune cells. The following stage is characterised by cellular proliferation and migration of various cell types which are involved in tissue formation, including fibroblasts which lay down initially disorganised extracellular matrix (ECM) components such as collagen. Deposition of collagen results in scar tissue formation, which is positively correlated to the duration of wound healing, particularly the inflammatory stage. The remodelling stage mainly involves remodelling of the ECM resulting in a more fibrous appearance to strengthen the repaired tissue (Gurtner et al. Wound Repair and Regeneration, Nature, 2008, 453: 314-321).

Platelets have a crucial role in wound healing. On activation in response to tissue damage, platelets release factors which trigger a coagulation cascade resulting in the deposition of fibrin and formation of a fibrin clot, which acts as a scaffold to trap cells and form a blood clot. The activated platelets also release growth factors and cytokines that stimulate cell migration, cell proliferation, angiogenesis, and inflammation. Thus, the platelets recruit immune cells and cells involved in wound healing to the site of injury, and trap them at the wound, and therefore initiate, promote, and sustain tissue repair.

Current biologic treatment strategies have not achieved tendon regeneration but include the use of ECM patches to provide a scaffold for new cell growth and differentiation (as reviewed in Galatz et al. Tendon Regeneration and Scar Formation: The Concept of Scarless Healing, J. Orthop. Res. 2015, 33(6) 823-831). Treatment modalities such as non-steroidal antiinflammatory drugs (NSAIDs) and corticosteroids can be used to treat tendinopathy and other disorders, but only offer symptomatic relief. Acute injury and chronic pathology often require surgical intervention, but surgical outcomes are unpredictable and often associated with persistent pain and discomfort. The poor self-repair capability of tendon and ligament tissue, and the limitations of current surgical and injection-based interventions have led to increased interest in platelet-rich plasma (PRP) (Chen et al. Am J Sports Med. 2018, 46(8): 2020-2032).

PRP comprises a mixture of highly concentrated platelets and associated growth factors produced by centrifugal separation of whole blood. PRP therapy is now widely used for treating musculoskeletal injuries (Zhou & Wang, PRP Treatment Efficacy for Tendinopathy: A Review of Basic Science Studies, BioMed. Res. Int. 2016). The main advantages of PRP that enable its widespread use are its safety and simple preparation and administration methods. Autologous PRP is safe because it is derived from a subject’s own blood and contains platelets and bioactive factors that can modify the biological microenvironment at injury sites, thus enhancing tissue healing. However, the efficacy of PRP for use in the treatment of connective tissue pathologies in clinical trials is still controversial. Studies using animal models, for example, have not conclusively shown that PRP affects tendon repair, which is consistent with the contradictory outcomes of the clinical use of PRP for managing tendon injury (Oh et al. Am J Sports Med. 2015, 11 (4):223-233).

There is, therefore, a need to provide more effective treatments for connective tissue injury, including ligament and tendon injury.

It has now been found that administration of vitamin A in combination with PRP surprisingly greatly improves healing of injured connective tissue, and had a dramatic and unexpected effect on healing of a reinjury. According to the invention, there is provided a method of treating a connective tissue injury in a subject comprising administering to the subject an effective amount of vitamin A and PRP.

There is also provided according to the invention vitamin A and PRP for use in the treatment of a connective tissue injury in a subject.

There is further provided according to the invention PRP for use in the treatment of a connective tissue injury in a subject administered vitamin A.

There is also provided according to the invention vitamin A for use in the treatment of a connective tissue injury in a subject administered PRP.

According to the invention there is also provided use of vitamin A and PRP in the manufacture of a medicament for the treatment of a connective tissue injury in a subject.

There is further provided according to the invention use of PRP in the manufacture of a medicament for the treatment of a connective tissue injury in a subject administered vitamin A.

There is also provided according to the invention use of vitamin A in the manufacture of a medicament for the treatment of a connective tissue injury in a subject administered PRP.

The term “connective tissue” is used herein to refer to several different tissues of the body that serve to connect, support and help bind other tissues in the body. Connective tissue can further be broken down into three categories: loose connective tissue, dense connective tissue, and specialized connective tissue. Loose connective tissue works to hold organs in place and is made up of extracellular matrix and collagenous, elastic and reticular fibers. Dense connective tissue is what makes up tendons and ligaments and consist of a higher density of collagen fibers. Examples of specialized connective tissues are adipose tissue, cartilage, bone, blood, and lymph. The injury may be any injury that causes damage to connective tissue, particularly dense connective tissue, such as ligament or tendon tissue. Such injuries may be caused, for example, by a strain, sprain, contusion, or a burn. The injury may, for example, be a sporting injury. The injury may be a reinjury.

Optionally the connective tissue injury is a dense connective tissue injury.

Optionally, the connective tissue injury is a tendon injury.

Optionally, the connective tissue injury is a ligament injury.

Optionally the subject is a human subject. According to clinical observations and statistical data, certain tendons are prone to a higher possibility of injury. These are the rotator cuff, forearm extensors, Achilles tendon, tibialis posterior and patellar tendons. Common ligament injuries include: knee ligament injuries, such as: anterior cruciate ligament (ACL) tear, medial cruciate ligament (MCL) or lateral cruciate ligament (LCL) sprain, Patella (knee cap) dislocation; ankle ligament injuries, such as: ankle sprain, achilles tear or rupture; shoulder ligament injuries, such as: shoulder dislocation, AC joint injury, rotator cuff tear; wrist and hand ligament injuries, such as: finger sprain or thumb sprain; spinal ligament injuries, such as: neck sprain, back ligament sprain, whiplash, text neck.

Optionally the subject is a horse.

Examples of tendon injuries common in horses (including in eventers, racehorses, polo horses) include injuries of the flexor tendons which are the deep digital flexor tendon (DDFT) and the superficial digital flexor tendon (SDFT) which run down the back of the limb from the level of the knee/hock. Common ligament injuries in the horse include: suspensory ligament desmitis, collateral ligaments of the coffin joint, fetlock joint and hock joints, palmar annular ligament of the fetlock, accessory (check) ligament of the deep flexor tendon, meniscal and cruciate ligaments of the stifle.

Any of these injuries may be treated in accordance with the invention.

The vitamin A and the PRP may be co-administered, or administered sequentially to the subject.

Optionally, vitamin A and the PRP are co-administered to the subject.

Optionally, the vitamin A and the PRP are administered sequentially to the subject.

The vitamin A and the PRP may be administered to the subject in any order. Optionally, the vitamin A is administered to the subject before the PRP. A plurality of doses of the vitamin A may be administered to the subject prior to administration of PRP.

Vitamin A

Vitamin A is the name of a group of fat-soluble retinoids, including retinol, retinal, and retinyl esters. There are two different categories of vitamin A. The first category, preformed vitamin A, comprises retinol and its esterified form, retinyl ester. The second category, provitamin A, comprises provitamin A carotenoids such as alpha-carotene, beta-carotene and betacryptoxanthin. Both retinyl esters and provitamin A carotenoids are converted to retinol, which is oxidized to retinal and then to retinoic acid. Both provitamin A and preformed vitamin A are known to be metabolized intracellularly to retinal and retinoic acid, the bioactive forms of vitamin A.

Vitamin A for use, or use of vitamin A, according to the invention may be an isolated form of vitamin A. An isolated form of vitamin A is any form of vitamin A found in the diet or a metabolized form thereof. For example, vitamin A may be isolated from fish liver oil. Vitamin A may comprise a preformed vitamin A such as retinol or a retinyl ester. Retinyl esters include retinyl acetate and retinyl palmitate. Vitamin A may comprise a provitamin A, such as a provitamin A carotenoid including alpha-carotene, beta-carotene or beta-cryptoxanthin. Vitamin A may comprise a bioactive form of vitamin A such as retinal or retinoic acid.

Vitamin A is available for human consumption in multivitamins and as a stand-alone supplement, often in the form of retinyl acetate or retinyl palmitate. A portion of the vitamin A in some supplements is in the form of beta-carotene and the remainder is preformed vitamin A; others contain only preformed vitamin A or only beta-carotene. Supplement labels usually indicate the percentage of each form of the vitamin. The amounts of vitamin A in stand-alone supplements range widely. Multivitamin supplements typically contain 2,500 to 10,000 international units (III) vitamin A, often in the form of both retinol and beta-carotene.

Vitamin A is listed on food and supplement labels in international units (ILIs). However, Recommended Dietary Allowance (RDA) (average daily level of intake sufficient to meet the nutrient requirements of nearly all (97%-98%) healthy individuals) for vitamin A is given as micrograms (pg; mcg) of retinol activity equivalents (RAE) to account for the different bioactivities of retinol and provitamin A carotenoids (see Table 1 below). Because the body converts all dietary sources of vitamin A into retinol, 1 mcg of physiologically available retinol is equivalent to the following amounts from dietary sources: 1 mcg of retinol, 12 mcg of betacarotene, and 24 mcg of alpha-carotene or beta-cryptoxanthin. From dietary supplements, the body converts 2 mcg of beta-carotene to 1 mcg of retinol.

Conversion rates between mcg RAE and IU are as follows:

• 1 IU retinol = 0.3 mcg RAE;

• 1 IU beta-carotene from dietary supplements = 0.15 mcg RAE;

• 1 IU beta-carotene from food = 0.05 mcg RAE; and

• 1 IU alpha-carotene or beta-cryptoxanthin = 0.025 mcg RAE.

An RAE cannot be directly converted into an IU without knowing the source(s) of vitamin A. For example, the RDA of 900 mcg RAE for adolescent and adult men is equivalent to 3,000 IU if the food or supplement source is preformed vitamin A (retinol). However, this RDA is also equivalent to 6,000 IU of beta-carotene from supplements, 18,000 IU of beta-carotene from food, or 36,000 IU of alpha-carotene or beta-cryptoxanthin from food. So a mixed diet containing 900 mcg RAE provides between 3,000 and 36,000 IU of vitamin A, depending on the foods consumed.

Table 1 : Recommended Dietary Allowances (RDAs) for Vitamin A

* Adequate Intake (Al), equivalent to the mean intake of vitamin A in healthy, breastfed infants.

Source: National Institutes of Health, Vitamin A, Fact Sheet for Health Professionals, as updated 5 October 2018

The Food and Nutrition Board (FNB) at the Institute of Medicine of the National Academies (formerly National Academy of Sciences) has established tolerable Upper Intake Level (UL) (maximum daily intake unlikely to cause adverse health effects) for preformed vitamin A that apply to both food and supplement intakes. The FNB based these ULs on the amounts associated with an increased risk of liver abnormalities in men and women, teratogenic effects, and a range of toxic effects in infants and children. The FNB has not established ULs for beta-carotene and other provitamin A carotenoids.

Table 2: Tolerable Upper Intake Levels (ULs) for Preformed Vitami

Age Male Female Pregnancy L mcg RAE 600 mcg RAE §

0 IU) (2,000 IU) §

L 600 mcg RAE 600 mcg RAE § p-d years |_{(2 0}00 IU) (2,000 IU) | |

o 900 mcg RAE 900 mcg RAE §

|4-a years _{(3 0}00 IU) |(3,000 IU) | |

L 1 ,700 mcg RAE 1 ,700 mcg RAE

§⁹-^{13 years} |(5,e®7 IU) 1(5,667 IU) | |

L_4-1O ,800 mcg RAE £,800 mcg RAE £,800 mcg RAE £,800 mcg RAE

P ^yearS |(9,333 IU) (9,333 IU) (9,333 IU) (9,333 IU) Table 2: Tolerable Upper Intake Levels (ULs) for Preformed Vitamin A*

Age Male Female Pregnancy Lactation

L _ 3,000 mcg RAE 3,000 mcg RAE 3,000 mcg RAE 3,000 mcg RAE y years |₍10,000 IU) (10,000 IU) |(¹°,°^{OO I U})

* These ULs, expressed in mcg and in lUs (where 1 mcg = 3.33 IU), only apply to products from animal sources and supplements whose vitamin A comes entirely from retinol or ester forms, such as retinyl palmitate. However, many dietary supplements (such as multivitamins) do not provide all of their vitamin A as retinol or its ester forms. For example, the vitamin A in some supplements consists partly or entirely of beta-carotene or other provitamin A carotenoids. In such cases, the percentage of retinol or retinyl ester in the supplement should be used to determine whether an individual’s vitamin A intake exceeds the UL. For example, a supplement labeled as containing 10,000 IU of vitamin A with 60% from beta-carotene (and therefore 40% from retinol or retinyl ester) provides 4,000 IU of preformed vitamin A. That amount is above the UL for children from birth to 13 years but below the UL for adolescents and adults.

The Vitamin A may be provided from a mixture of different sources, including, for example, in normal feed, and in the form of a supplement.

It will be appreciated that use of a natural vitamin for tissue repair is particularly advantageous because of its known safety profile.

A maximum safe dose of vitamin A (in particular of preformed vitamin A) per day for an adult human subject may be 100,000 IU vitamin A (in particular of preformed vitamin A). Vitamin A toxicity levels have been recorded at around 1 ,000 lU/kg body weight (BW)/day for a horse. Thus, a maximum safe dose of vitamin A (in particular of preformed vitamin A) per day for an adult horse subject may be 1 ,000 lU/kg BW vitamin A (in particular of preformed vitamin A).

Optionally the subject is administered up to 50% (for example >10% to 50%, or 25% to 50%) of a maximum safe dose of vitamin A (in particular of preformed vitamin A) for the subject per day.

Optionally the subject may be administered a dose of vitamin A which is up to 50% of a minimum toxic dose for the subject. Optionally the subject may be administered a dose of vitamin A which is at least 5% of a minimum toxic dose for the subject.

For example, a minimum toxic dose for a subject may be 1 ,000 lll/kg body weight (BW)/day.

Optionally the subject is a human subject.

Optionally for an adult human subject (>18 years old), the subject may be administered up to 100,000 III vitamin A (in particular of preformed vitamin A) per day. For example, >10,000 IU to 100,000 IU vitamin A per day; about 25,000 to 100,000 IU vitamin A per day; about 50,000 to 100,000 IU vitamin A per day; or about 75,000 to 100,000 IU vitamin A per day (in particular of preformed vitamin A).

Optionally for an adult human subject (>18 years old), the subject may be administered up to 25,000 IU vitamin A per day (in particular of preformed vitamin A). For example, >10,000 IU to 25,000 IU vitamin A per day (in particular of preformed vitamin A).

Optionally the subject is a horse.

Optionally a horse may be administered a dose of 25,000-250,000, 50,000-250,000, 75,000- 250,000, 100,000-250,000, 125,000-250,000, 150,000-250,000, 175,000-250,000, or 200,000-250,000 IU vitamin A per day.

Optionally a horse may be administered a dose of 25,000-50,000, 25,000-75,000, 25,000- 100,000, 25,000-125,000, 25,000-150,000, 25,000-175,000, or 25,000-200,000 IU vitamin A per day.

The vitamin A may be administered to a subject systemically for example orally or intravenously.

Vitamin A may be administered to the subject at least once per day, twice per day, three times per day, four times per day, or five times per day.

Vitamin A may be administered to the subject at least once per day for at least 3 days for example for at least a week, for at least a month, or for at least 6 months from the day of first administration to the subject.

Prolonged exposure to high doses of vitamin A may lead to hypervitaminosis A. Thus, it may be preferred to limit administration of high doses of vitamin A to the subject for up to 6 years, or up to 6 months, from the day of first administration to the subject.

It will be appreciated that vitamin A should preferably be administered as soon as possible after an injury has occurred. Optionally vitamin A is first administered within a day, a week, or a month of the injury. Optionally vitamin A is first administered within a week of the injury. However, beneficial effects of treatment with vitamin A may be observed when treatment is initiated many months or even years after an injury has occurred. Thus, optionally vitamin A may be administered months, years or even decades after an injury has occurred, for example within six months, a year, or a decade, or within twenty, thirty, forty, fifty, or sixty years of the injury.

Treatment with vitamin A may begin before surgery to repair the injured tissue.

Vitamin A treatment may begin after surgery to repair the injured tissue.

Vitamin A treatment may begin before surgery and be continued after surgery to repair the injured tissue.

Maintenance doses of vitamin A

We have found that administering a post- treatment maintenance dose of vitamin A supplement provides continued benefits, especially for treatment of connective tissue injuries. As described in more detail in Example 2 below, after several weeks of treatment with “full-treatment doses” of vitamin A supplement, horses with connective tissue injuries (tendon or ligament) were orally administered vitamin A once per day with a reduced, “maintenance dose” of half a “full-treatment dose” of vitamin A supplement for 7 weeks. The results provide evidence for beneficial effects of continuing with a maintenance dose of vitamin A supplement after a period of administration of full-treatment doses.

Accordingly, after a period of treatment of a subject with “full-treatment doses” of Vitamin A, it may be advantageous to continue treatment for a further period with “maintenance doses” of Vitamin A.

In one embodiment, one or more maintenance doses of vitamin A are administered to the subject.

A “maintenance dose” is a dose of vitamin A, or of a composition comprising vitamin A, which is less than a “full-treatment dose”.

Optionally a maintenance dose is up to three quarters of a full-treatment dose, or up to two- thirds of a full treatment dose.

Optionally a maintenance dose is at least a quarter of a full-treatment dose.

Optionally a maintenance dose is administered to the subject after the subject has been administered one or more full-treatment doses. Optionally a maintenance dose is administered to the subject from the day after the last administration of a full-treatment dose.

Typically, a plurality of maintenance doses are administered to the subject.

Optionally the maintenance doses are administered for at least one week from the day of first administration of a maintenance dose to the subject.

Optionally the maintenance doses are administered for at least four weeks from the day of first administration of a maintenance dose to the subject.

Optionally the maintenance doses are administered for at least 12 weeks from the day of first administration of a maintenance dose to the subject.

Optionally the maintenance doses are administered for at least 6 months from the day of first administration of a maintenance dose to the subject.

Optionally the maintenance doses are administered for at least 12 months from the day of first administration of a maintenance dose to the subject.

Optionally the maintenance doses are administered for up to 6 years from the day of first administration of a maintenance dose to the subject.

Optionally, the PRP is administered at least one week after the last administration of a maintenance dose of vitamin A to the subject.

Optionally the subject is a human subject.

For a human subject the, or each full-treatment dose optionally comprises >10,000 to 100,000 III vitamin A per day.

For a human subject the, or each full-treatment dose optionally comprises about 25,000-

100,000, 50,000-100,000, or 75,000-100,000 IU vitamin A per day

For a human subject the, or each maintenance dose optionally comprises >2,500 IU to 75,000 IU vitamin A per day.

For a human subject optionally the, or each maintenance dose comprises 5,000-75,000, 10,000-75,000, or 20,000-75,000 IU vitamin A per day.

For a human subject optionally the, or each maintenance dose comprises 5,000-50,000, 10,000-50,000, or 20,000-50,000 IU vitamin A per day.

Optionally the subject is a non-human subject.

Optionally the subject is a horse. For a horse, optionally the, or each full-treatment dose comprises 25,000-250,000, 50,000- 250,000, 75,000-250,000, 100,000-250,000, 125,000-250,000, 150,000-250,000, 175,000- 250,000, or 200,000-250,000 III vitamin A per day.

For a horse, optionally the, or each maintenance dose comprises 10,000-200,000, 20,000- 150,000, 40,000-100,000, or 60,000-100,000 IU vitamin A per day.

A “maintenance dose” may be administered as a single dose, or in multiple dose units. For example, a maintenance dose of 80,000 IU vitamin A per day for a horse may be provided as two doses of 40,000 IU vitamin A, one dose to be given in the morning, and another dose to be given in the evening.

Similarly, a “full-treatment dose” may be administered as a single dose, or in multiple dose units. For example, a full-treatment dose of 160,000 IU vitamin A per day for a horse may be provided as two doses of 80,000 IU vitamin A, one dose to be given in the morning, and another dose to be given in the evening.

The vitamin A can be incorporated into a variety of formulations for therapeutic administration, more particularly by combination with appropriate, pharmaceutically acceptable carriers, pharmaceutically acceptable diluents, or other pharmaceutically acceptable excipients, and can be formulated into preparations in solid, semi solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols as appropriate.

Optionally the vitamin A is in solid form.

Optionally the vitamin A is not in an organic solution.

Optionally the vitamin A is not encapsulated by, or attached to a microparticle.

Optionally the vitamin A is not encapsulated by, or attached to a nanoparticle.

Vitamin A can be administered in the form of a pharmaceutically acceptable salt. It can also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. Optionally vitamin A is administered with an antibiotic agent. Optionally vitamin A is the only non-cellular, non-antibiotic, active agent administered.

The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, vitamin A can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavouring agents.

Vitamin A can be formulated into preparations for injection by dissolving, suspending or emulsifying in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, propylene glycol, synthetic aliphatic acid glycerides, injectable organic esters (e.g., ethyl oleate), esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Furthermore, a pharmaceutical composition of the present disclosure can comprise further agents such as dopamine or psychopharmacologic drugs, depending on the intended use of the pharmaceutical composition.

Pharmaceutical compositions are prepared by mixing Vitamin A having the desired degree of purity with optional physiologically acceptable carriers, other excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, other excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

The pharmaceutical composition can be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents can be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

An aqueous formulation can be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate- buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

A tonicity agent can be included in the formulation to modulate the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions can be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as a physiological salt solution or serum. Tonicity agents can be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 nM.

A surfactant can also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactant can range from about 0.001% to about 1 % w/v.

A lyoprotectant can also be added in order to protect a labile active ingredient against destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 nM.

In some embodiments, a subject formulation includes one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, for example, at concentrations ranging from about 0.001 to about 2% (w/v).

Platelet-rich plasma (PRP)

PRP is obtained by centrifuging whole blood, and comprises platelets, plasma and various white blood cells (WBCs). The normal platelet count in whole blood of a human subject is 150,000/pl to 400,000/pl (Halpern et al. “The Role of Platelet-Rich Plasma in Inducing Musculoskeletal Tissue Healing", Hospital For Special Surgery. 2012, 8:137-145). By definition, PRP contains a higher concentration of platelets than baseline levels in whole blood, however in clinical use PRP may have a platelet concentration much higher than that of whole blood, the concentration depending on its application (Yuan et al. “Augmenting tendon and ligament repair with platelet-rich plasma”, Muscles, Ligaments and Tendons Journal. 2013, 3(3): 139- 149). A study conducted to look into the effect of PRP releasate (PRPr) on the early stages of tendon healing found that PRPr from roughly 3,500,000 platelets/pL (3.4 fold higher than the platelet count of whole rat blood, which is normally 900,000/pL) resulted in the promotion of tissue recovery in the early phase of Achilles tendon healing in rats (Yu et al. Platelet-Rich Plasma Releasate Promotes Early Healing in Tendon After Acute Injury. Journal of Experimental Orthopaedics. 2021 , https://doi.orq/10.1177%2F2325967121990377). A further study looking at the efficacy of PRP on treating murine patellar tendon injury found that roughly 7,000,000 platelets/pL (7.1- fold higher platelet count than whole murine blood, which is normally 900,000 to 1 ,600,000/pL) resulted in significantly faster tendon healing than a control group (Kobayashi et al. PRP Accelerates Murine Patellar Tendon Healing Through Enhancement of Angiogenesis and Collagen Synthesis. Journal of Experimental Orthopaedics. 2020, 7, 49)). In vitro studies on human tenocytes cultured with PRP show that platelet concentrations of 500,000-2,000,000 platelets/pL result in increased tenocyte proliferation, migration, and collagen and MMP production compared to control tenocytes not cultured with PRP (Giusti et al. “Platelet Concentration in Platelet-Rich Plasma Affects Tenocyte Behaviour In Vitro”. BioMed Research International. 2014, doi: 10.1155/2014/630870).

As shown from these studies, a platelet count of three to five times the baseline of a particular species, or at least 1 ,000,000/pl is typically used (Halpern et al, supra), however higher platelet counts, for example, of 7-fold or more, may also be used.

Optionally the platelet concentration of the PRP comprises at least 500,000 platelets/pl.

Optionally the platelet concentration of the PRP comprises at least 1 ,000,000 platelets/pl. Optionally the platelet concentration of the PRP comprises at least 1 ,500,000 platelets/pl.

Platelets contain a-granules that contain proteins, cytokines, growth factors and other bioactive factors that initiate and regulate wound healing. Platelet life span is 5-9 days, wherein they continually regenerate growth factors and other active agents after release of the a-granule content.

Growth factors present in PRP include, in particular, transforming growth factor (TGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived endothelial growth factor (PDEGF), platelet-derived angiogenesis factor (PDAF), platelet factor 4 (PF-4), hepatocyte growth factor (HGF).

Multiple commercial PRP products are available, and deliver different concentrations of platelets, red blood cells (RBCs), and WBCs (see, for example, as described in Halpern et al., Hospital for Special Surgery 2012, 8:137-145). PRP products include platelet-rich concentrate (pure PRP), platelet gel (PG), platelet releasate, and isolated growth factors. PG are blood-derived biomaterials that are generally obtained through the activation of PRP or a platelet concentrate by thrombin or calcium chloride, resulting in the simultaneous conversion of fibrinogen into a fibrin gel and in the generation of a platelet releasate rich in a physiological cocktail of growth factors (T. Burnouf. “Platelet Gels". ISBT Science Series (2013), 8, 131-136). Platelet releasate (PRPr) is a cell-free preparation of active agents released from platelets. Several commercially available PRP systems and their PRP preparations are described in Le et al., “Current Clinical Recommendations for Use of Platelet-Rich Plasma”, Current Reviews in Musculoskeletal Medicine (2018) 11 :624-634.

The optimum concentration of PRP varies for treatment of different tissue types. Increased WBCs can enhance further tissue repair due to cytokines contained in WBCs, and enhance the antimicrobial effect of PRP, but they may also increase local inflammation and may, therefore, impede tissue recovery. The effect of WBCs within a PRP preparation is likely to be tissue specific. RBC presence in the PRP matrix may have an irritant effect due to the haem in the RBCs; thus, fewer RBCs in the mix may allow less pain after injection. Platelet activation and growth factor release occurs with the addition of calcium or thrombin or the exposure to collagen. In this way, the platelets can be activated with calcium or thrombin just prior to injection or once injected into the patient by activation with the patient’s own collagen to then release the growth factors. There are, therefore, time restraints when using PRP in order to avoid premature activation far in advance of injecting the patient. The choice of platelet activator will affect the growth factor release profile from platelets. Thrombin activated platelets release growth factors immediately, whereas collagen activation of PRP results in a slower sustained release over 5 days. The effect of PRP depends on the inflammatory cascade, and so anti-inflammatory drugs (such as NSAIDS) may be administered before andafter PRP administration (for example, one week before, and two weeks after PRP administration).

A convenient source of PRP is from blood extracted from the subject to be treated. PRP may thus be obtained and activated for use on the same subject. Methods of using a subject's own blood are called "autologous" or "autogenic" donor methods. When the blood is donated by a subject of the same species (but a different individual) as the subject to be treated, this is termed a "homologous" source. Homologous sources of PRP are less preferred as they may potentially be biologically or immunologically incompatible with the subject and there is a potential risk of contamination with infectious agents, such as hepatitis and HIV contaminants. Autologous PRP is preferred as it has several safety advantages. For example, since PRP is generally a by-product of the subject’s own blood, disease transmission or immunological reactions are not an issue. However, for example, if the subject suffers with a complex systemic disease, this can affect the concentration of growth factors in their blood and in any of the blood-derived preparations. Such subjects, therefore, may not benefit from the advantages of this type of treatment.

In some circumstances (such as those described above), PRP sourced from another species ("heterologous" PRP) may be used since this can provide a reliable, readily available, and highly reproducible source of raw material. For example, porcine blood may be used for human subjects as it is less likely to carry a human viral infectious risk (HIV, hepatitis, etc), and porcine and human growth factors are nearly identical to each other .

Without being bound by theory, it is believed that PRP re-starts the wound healing process (beginning with an inflammatory cascade, followed by proliferation/regeneration and remodelling phases), and effectively turns a chronic injury into an acute injury.

Optionally PRP is first administered to the subject within twelve months of the injury.

Optionally PRP is first administered to the subject within six months of the injury.

Optionally PRP is first administered to the subject within three months of the injury.

Optionally PRP is first administered to the subject within a month of the injury.

Optionally PRP is first administered to the subject within twelve months of first administration of vitamin A to the subject.

Optionally PRP is first administered to the subject within six months of first administration of vitamin A to the subject. Optionally PRP is first administered to the subject within three months of first administration of vitamin A to the subject.

Optionally PRP is first administered to the subject within a month of first administration of vitamin A to the subject.

Optionally PRP is first administered to the subject within three weeks of first administration of vitamin A to the subject.

Echogenicity is a measure of the ability of a surface to reflect ultrasound. Structures composed of different tissue will have different echogenicities. The health of an injured tissue, such as a tendon or a ligament, can be assessed by comparing the echogenicity of the injured tissue with that of a corresponding healthy tissue. Healthy tendons comprising uninjured tissue and normal architecture are usually hyperechoic and appear white on an ultrasonogram; they are capable of reflecting ultrasound that is cast over the tissue. An injured tendon comprising a lesion will appear as less hyperechoic, and more hypoechoic as tendon fibers are interrupted and defects are filled with fluid, blood, or fat. Severe lesions will be anechoic, and will display as completely dark on an ultrasonogram.

Methods for determining echogenicity of tendons and ligaments are described in Example 1 below and, for example, in:

Spinella et al., “Relative Echogenicity of Tendons and Ligaments in Foals", PLOS ONE, DOI:10.1371/journal. pone.0159953, July 21 , 2016;

Smith et al., “Ultrasonography of Tendon and Ligament Injury - New Techniques for an Established Imaging Modality”, 2012 NAVC Proceedings, March 8, 2019; and

Schmidt et al, “Ultrasound Echogenicity is Associated with Achilles Tendon Fatigue Damage in a Cadaveric Loading Model”. bioRxiv, doi: November

21 , 2019.

A ratio of echogenicity of an injured tissue, such as a tendon or ligament, to echogenicity of a corresponding healthy tissue can be used to provide a measure of the progress of wound healing. An echogenicity ratio of 1 :1 (injured: healthy tissue) indicates full regeneration of an injured tendon. This may be determined by any suitable method, for example as described below in Example 1. An echogenicity ratio of lesion:healthy tissue may be used to provide an assessment of the echogenicity of the main lesion.

Optionally, PRP is administered to the subject once a ratio of echogenicity of injured connective tissue of the connective tissue injury to echogenicity of corresponding uninjured (healthy) connective tissue is greater than 0.6. Optionally, PRP is administered to the subject once a ratio of echogenicity of injured connective tissue of the connective tissue injury to echogenicity of corresponding uninjured (healthy) connective tissue is greater than 0.7.

Optionally, PRP is administered to the subject once a ratio of echogenicity of injured connective tissue of the connective tissue injury to echogenicity of corresponding uninjured (healthy) connective tissue is greater than 0.8.

For example, in horses, corresponding uninjured (healthy) tissue may be an adjacent healthy tendon to an injured tendon.

Optionally the subject is administered a dose of PRP wherein the platelet concentration is at least 500,000 platelets/pl.

Optionally the subject is administered a dose of PRP wherein the platelet concentration is at least 1 ,000,000 platelets/pl.

Optionally the subject is administered a dose of PRP wherein the platelet concentration is at least 1 ,500,000 platelets/pl.

It will be appreciated that an effective amount of PRP may be selected depending on the subject, and determined depending on the severity of disease, a patient's age, body weight, health conditions, gender, and drug sensitivity, administration time, administration route, excretion rate, treatment period, and drugs blended with or co-administered with the PRP, and other factors well known in the medical field.

PRP may be administered to the subject by any suitable route, including for example by injection directly into the injured connective tissue (such as intratendinous injection, or intraligament injection), by intra-articular injection (directly into an injured joint), by extra- articular injection, or by intravenous injection. Optionally PRP is administered to the subject intravenously.

A plurality of doses of PRP may be administered to a subject in need thereof. Each dose of PRP may be administered via the same or a different route as the initial administration.

Optionally PRP is administered to the subject at least once every month.

Optionally PRP is administered to the subject at least once every six months.

Optionally a second administration of PRP may be administered up to one year later than a first administration of PRP. For example, a second administration of PRP may be up to six months later than a first PRP administration, up to three months later than the first PRP administration, or up to one month later than the first PRP administration. A second administration of PRP may comprise a dose with fewer platelets than the first administration.

The PRP to be administered to the subject may comprise platelets obtained from the subject.

Optionally the PRP is autologous PRP. For example, the PRP may be prepared from blood collected from a subject, and then the prepared PRP may be administered to the same subject.

Optionally the PRP is homologous PRP.

Optionally the PRP is heterologous PRP.

PRP may be administered to the subject in one or more unit doses.

The PRP may also comprise a platelet activator. The platelet activator may coagulate PRP. For example, the platelet activator may be thrombin, calcium chloride, calcium gluconate, or a combination thereof. The platelet activator and the PRP may be administered at the same time or separately.

Alternatively, the PRP may not comprise a platelet activator. The activation of platelets in the PRP may be dependent on the subject’s own platelet activators, such as collagen.

Growth factors released by activated platelets play a crucial role in enhancing wound healing. In addition to the growth factors contained in the a-granules of platelets, the PRP may also comprise additional growth factors and other active agents. In particular, the PRP may additionally comprise growth factors selected from the group consisting of transforming growth factors (TGF), fibroblast growth factors (FGF), platelet-derived growth factors (PDGF), epidermal growth factors (EGF), vascular endothelial growth factors (VEGF), insulin-like growth factors (IGF), platelet-derived endothelial growth factors (PDEGF), platelet-derived angiogenesis factors (PDAF), platelet factors 4 (PF-4), hepatocyte growth factors (HGF) and mixtures thereof.

The PRP may further include a carrier, an excipient, or a diluent, such as, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oils or a combination thereof.

Combined Preparations and Pharmaceutical Compositions

There is also provided according to the invention a combined preparation, which comprises: (a) vitamin A; and (b) platelet-rich plasma (PRP). Optionally the vitamin A of a combined preparation of the invention is separate from the PRP.

The term "combined preparation" as used herein refers to a "kit of parts" in the sense that the combination components (a) and (b) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination components (a) and (b). The components can be administered simultaneously or one after the other. If the components are administered one after the other, preferably the time interval between administration is chosen such that the therapeutic effect of the combined use of the components is greater than the effect which would be obtained by use of only any one of the combination components (a) and (b).

The components of the combined preparation may be present in one combined unit dosage form, or as a first unit dosage form of component (a) and a separate, second unit dosage form of component (b). The ratio of the total amounts of the combination component (a) to the combination component (b) to be administered in the combined preparation can be varied, for example in order to cope with the needs of a patient sub-population to be treated, or the needs of the single patient, which can be due, for example, to the particular disease, age, sex, or body weight of the patient.

Preferably, there is at least one beneficial effect, for example an enhancing of the effect of the vitamin A, or an enhancing of the effect of the PRP, or a mutual enhancing of the effect of the combination components (a) and (b), for example a more than additive effect, additional advantageous effects, fewer side effects, less toxicity, or a combined therapeutic effect compared with an effective dosage of one or both of the combination components (a) and (b), and very preferably a synergism of the combination components (a) and (b).

There is also provided according to the invention a pharmaceutical composition, which comprises: (a) vitamin A; (b) PRP; and (c) a pharmaceutically acceptable carrier, excipient, or diluent.

Optionally the vitamin A and the PRP are for co-administration to a subject.

Optionally the vitamin A and the PRP are for sequential administration to a subject.

Optionally a combined preparation or a pharmaceutical composition of the invention comprises a plurality of doses of the vitamin A.

Optionally a combined preparation or a pharmaceutical composition of the invention comprises a plurality of doses of the PRP.

Optionally the vitamin A comprises isolated vitamin A.

Optionally the vitamin A comprises a preformed vitamin A, such as a retinyl ester or retinol. Optionally the vitamin A comprises a provitamin A, such as a carotenoid.

Optionally the vitamin A comprises a bioactive form of vitamin A, such as retinal or retinoic acid.

Optionally the vitamin A is present in a unit dose form which comprises up to 50% of a maximum safe daily dose of vitamin A for a subject per day.

Optionally the vitamin A is present in a unit dose form which comprises >10% to 50% of a maximum safe daily dose of vitamin A for a subject.

Optionally the vitamin A is present in a unit dose form which comprises 25% to 50% of a maximum safe daily dose of vitamin A for a subject.

Optionally the vitamin A is present in a unit dose form which comprises up to 50% of a minimum toxic dose for a subject.

Optionally the vitamin A is present in a unit dose form which comprises at least 5% of a minimum toxic dose for a subject.

Optionally the subject is a human subject.

Optionally, for a human subject, the vitamin A is present in a unit dose form which comprises >10,000 to 100,000 IU vitamin A.

Optionally, for a human subject, the vitamin A is present in a unit dose form which comprises about 25,000-100,000, 50,000-100,000, or 75,000-100,000 IU vitamin A.

Optionally the subject is a horse.

Optionally, for a horse, the vitamin A is present in a unit dose form which comprises 25,000- 250,000, 50,000-250,000, 75,000-250,000, 100,000-250,000, 125,000-250,000, 150,000- 250,000, 175,000-250,000, or 200,000-250,000 IU vitamin A.

Optionally, for a horse, the vitamin A is present in a unit dose form which comprises 25,000- 50,000, 25,000-75,000, 25,000-100,000, 25,000-125,000, 25,000-150,000, 25,000-175,000, or 25,000-200,000 IU vitamin A.

Optionally the vitamin A is for systemic administration to a subject.

Optionally the vitamin A is for oral or intravenous administration to a subject.

Optionally the PRP is present in a unit dose form which comprises a platelet concentration of at least 500,000 platelets/pl. Optionally the PRP is present in a unit dose form which comprises a platelet concentration of at least 1 ,000,000 platelets/pl.

Optionally the PRP is present in a unit dose form which comprises a platelet concentration of at least 1 ,500,000 platelets/pl.

Optionally the PRP is for intravenous administration to a subject.

Optionally the PRP comprises autologous PRP.

Optionally the PRP comprises additional growth factors.

Optionally the vitamin A is present in a combined preparation or in a pharmaceutical composition of the invention in unit dose form which is a maintenance dose, which is less than a full treatment dose.

Optionally the maintenance dose is up to three quarters of a full treatment dose.

Optionally the maintenance dose is up to two-thirds of a full treatment dose.

Optionally the maintenance dose is at least a quarter of a full treatment dose.

Optionally the subject is a human subject.

Optionally, for a human subject, the, or each maintenance dose comprises >2,500 III to 75,000 IU vitamin A.

Optionally, for a human subject, the, or each maintenance dose comprises 5,000-75,000, 10,000-75,000, or 20,000-75,000 IU vitamin A.

Optionally, for a human subject, the, or each maintenance dose comprises 5,000-50,000, 10,000-50,000, or 20,000-50,000 IU vitamin A.

Optionally, the subject is a non-human subject.

Optionally the subject is a horse.

Optionally, for a horse, the, or each maintenance dose comprises 10,000-200,000, 20,000- 150,000, 40,000-100,000, or 60,000-100,000 IU vitamin A.

There is also provided according to the invention a combined preparation or a pharmaceutical composition of the invention for use as a medicament.

There is also provided according to the invention a combined preparation or a pharmaceutical composition of the invention for use in the treatment of a connective tissue injury. There is further provided according to the invention use of a combined preparation or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a connective tissue injury.

Unit dosages

Unit dosage (or unit dose) forms for oral administration such as syrups, elixirs, and suspensions can be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, or tablet contains a predetermined amount of the active agent (e.g. vitamin A). Similarly, unit dosage forms for injection or intravenous administration can comprise vitamin A in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier, or PRP with a suitable carrier, excipient or diluent (for example, as detailed above).

The term “unit dosage form,” (or “unit dose”), as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of vitamin A or PRP calculated in an amount sufficient to produce the desired effect, typically in association with a pharmaceutically acceptable diluent, carrier or vehicle. In some cases, two or more individual unit doses in combination provide a therapeutically effective amount of the active ingredient, for example, two tablets or capsules taken together (or sequentially) may provide a therapeutically effective dose, such that the unit dose in each tablet or capsule is approximately 50% of the therapeutically effective amount.

Combined preparations and pharmaceutical compositions of the invention may be packaged with instructions for administration of the components on the combination, or the composition. The instructions may be recorded on a suitable recording medium or substrate. For example, the instructions may be printed on a substrate, such as paper or plastic. The instructions may be present as a package insert, in the labeling of the container or components thereof (i.e., associated with the packaging or sub-packaging). In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, for example, CD-ROM, diskette. Some or all components of the combined preparation or pharmaceutical composition may be packaged in suitable packaging to maintain sterility.

Vitamin A can be administered as an injectable formulation. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle can contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of vitamin A and/or PRP adequate to achieve the desired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Ranges may be expressed herein as from “about” one particular value, and/or to another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about”, it will be understood that the particular value forms another embodiment.

Wherever the term “vitamin A” is used herein this includes reference to “vitamin A or a pharmaceutically acceptable salt thereof”.

Embodiments of the invention are described below, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 shows a line graph depicting lesion size (as a percentage of baseline lesion size) and echogenicity values (blue line is ratio of echogenicity of lesion:healthy corresponding tendon, red line is ratio of injured tendon:healthy corresponding tissue) in an injured equine tendon at specified time points after initial injury. Full dose of vitamin A commenced at week 0, which was replaced with maintenance dose at week 7, at week 8 the horse was put back on full-dose vitamin A before commencing PRP treatment at week 11.

Figure 2 shows a line graph depicting the effect of post-treatment maintenance dose of vitamin A supplement on the size of tendon/ligamentous lesions: (a) shows mean lesion size from week 0 to week 7 on full-treatment doses of vitamin A supplement before splitting into the values of the maintenance dose and placebo groups for week 7 to week 14; and (b) shows the lesion size for both maintenance and placebo groups from week 0 through to week 14, as well as the mean values for the groups from week 0 to week 7; Figure 3 shows a series of cross-sectional ultrasonograms of a tendon injury that became re-injured after administration of full-treatment doses of vitamin A supplement was stopped and a maintenance dose of supplement was administered. Figure (a) shows an ultrasonogram of the lesion at baseline (week 0) before treatment with vitamin A supplement commenced; (b) shows an ultrasonogram at week 7 of administration with full-treatment doses of vitamin A supplement; and (c) shows ultrasonogram of the lesion at week 14 (after 7 weeks of post full-treatment maintenance doses of vitamin A supplement); and

Figure 4 shows a bar graph depicting the effect of activity level on connective tissue improvement in horses on vitamin A supplement. Improvement is shown as percentage improvement from baseline. Data are presented as mean values, with error bars representing the standard error of the mean.

Figure 5(a) shows a cross-sectional ultrasonogram of the major tendons/ligaments present in the left foot of a horse (normal equine anatomy). Figure 5(b) shows a longitudinal ultrasonogram (left) of the left foot of a horse compared with a cross-sectional ultrasonogram (right) of the left foot of the same horse (normal equine anatomy);

Figure 6(a) shows a bar graph depicting size of lesion (as a % of baseline lesion size) in tendon/ligament at specified time points after commencing supplementation. Data are presented as mean values, with error bars representing the standard error of the mean. Statistically significant results (p < 0.05) compared to baseline were noted with an asterisk (*). Week 3: p = 0.169; Week 5: p = <0.001 ; Week 7: p = 0.006. Figure 6(b) shows scatter plots of percentage improvement (%) against time since injury (months) with all data points included (Pearson’s correlation p = 0.027);

Figure 7 shows a cross-sectional ultrasonogram of a lesion in the left forelimb check ligament in a horse (ID 1431): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks. There is movement artefact shown in the figure;

Figure 8 shows a cross-sectional ultrasonogram of a lesion in the left forelimb SDFT in a horse (ID 8827): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks;

Figure 9 shows a cross-sectional ultrasonogram of a lesion in the left hindlimb medial suspensory branch ligament in a horse (ID 10520): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks, (d) shows improvements in axial aspect of the branch;

Figure 10 shows a cross-sectional ultrasonogram of a lesion in the left forelimb SDFT in a horse (ID 111112): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks. Some filling in of lesion in lateral aspect of tendon;

Figure 11 shows a cross-sectional ultrasonogram of a lesion in the left forelimb SDFT in a horse (ID 123345): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks;

Figure 12 shows a cross-sectional ultrasonogram of a lesion in the right forelimb lateral SDFT in a horse (ID 1234567): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks. Tendon is clearly filling in well;

Figure 13 shows a cross-sectional ultrasonogram of a lesion in the left forelimb SDFT in a horse (ID Q1Q): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks; Figure 14 shows a cross-sectional ultrasonogram of a lesion in the left forelimb SDFT in a horse (ID REG6): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks;

Figure 15 shows a longitudinal ultrasonogram of a lesion in the right forelimb lateral suspensory branch ligament in a horse (ID REG9): (a) before administration of any vitamin A supplement comprising vitamin A; (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks; (c) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks; and (d) after daily administration of a pharmaceutical composition comprising vitamin A for 7 weeks. Image at week 7 (Figure 15(d)) shows improvement of injury as deeper lesion less visible.

Figure 16 shows a cross-sectional ultrasonogram of a lesion in the left forelimb check ligament in a horse (ID 1833): (a) before administration of any vitamin A supplement comprising vitamin A; and (b) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks. Some improvement of check ligament injury appearance. The horse also received platelet rich plasma;

Figure 17 shows a cross-sectional ultrasonogram of a lesion in the right forelimb check ligament in a horse (ID 1833): (a) before administration of any vitamin A supplement comprising vitamin A; and (b) after daily administration of a pharmaceutical composition comprising vitamin A for 5 weeks;

Figure 18 shows a cross-sectional ultrasonogram of a lesion in the left forelimb lateral SDFT in a horse (ID 6168): (a) before administration of any vitamin A supplement comprising vitamin A; and (b) after daily administration of a pharmaceutical composition comprising vitamin A for 3 weeks. The figure shows good infilling of lateral SDFT lesion.

Figure 19 shows line graphs depicting size of lesion (as a % of baseline lesion size) in tendon/ligament injuries at specified time points after original injury (OG): (a) shows tendon/ligaments as a single cohort; (b) shows tendon injuries as a separate, single cohort; (c) shows ligamentous injuries as a separate, single cohort; and (d) shows ligamentous injuries as a separate, single cohort with outlier removed from data set. Data are presented as actual values for each subject; Figure 20(a) shows a line graph depicting the mean echogenicity ratio of a tendon lesion to an adjacent healthy tendon at specified time points after commencing supplementation. Data are presented as mean values for the tissues, with error bars representing the standard error of the mean. A value of 1 would indicate perfect regeneration of native tendon. Figure 20(b) shows a cross-sectional ultrasonogram showing the outline of the tendon lesion of (a) with the injured tendon also outlined. Figure 20(c) shows a cross- sectional ultrasonogram showing the outline of an adjacent healthy tendon used as a comparison tissue to calculate the echogenicity ratio, as well as the outline of the lesion and injured tendon;

Figure 21(a) shows a line graph depicting the mean echogenicity ratio of an injured tendon (with the area of lesion excluded) to an adjacent healthy tendon at specified time points after commencing supplementation. Data are presented as mean values for the tissues, with error bars representing the standard error of the mean. A value of 1 would indicate perfect regeneration of native tendon. Figure 21(b) shows a cross-sectional ultrasonogram showing the outline of the tendon lesion of (a) with the injured tendon also outlined, the lesion is excluded from the area of injured tendon for analysis. Figure 21(c) shows a cross- sectional ultrasonogram showing the outline of an adjacent healthy tendon used as a comparison tissue to calculate the echogenicity ratio, as well as the outline of the lesion and injured tendon;

Example 1

Treatment of equine connective tissue injury with vitamin A supplement and platelet-rich plasma (PRP)

This example describes the effect of a combination therapy of vitamin A supplement with PRP in treating equine connective tissue injury.

Vitamin A supplement used:

Vitamin A palmitate (retinyl palmitate) in a vehicle, delivered in dry feed at 16,000 III per kg feed dry matter (full-dose treatment) or 8,000 IU per kg feed dry matter (maintenance dose treatment).

Administration of vitamin A supplement:

Full-dose treatment: oral administration of vitamin A palmitate in a vehicle, at a dose of 160,000 IU vitamin A once per day. Maintenance dose treatment: oral administration of vitamin A palmitate in a vehicle, at a dose of 80,000 III vitamin A once per day.

Methodology:

Rania (a female horse) sustained a tendon injury in April 2021 and was started on full-dose treatment with vitamin A for 7 weeks. She improved tremendously well in both lesion size and echogenicity, achieving nearly full resolution of her injury. After her initial treatment period of 7 weeks, she was administered a maintenance dose (half-dose) of vitamin A. Almost immediately after halving her dose, in week 8, she sustained an horrendous reinjury, and was placed back on full-dose treatment with vitamin A. Rania was administered PRP at week 11. She was box rested from when she sustained her original injury, but started full work from week 20.

Scans were taken at the following time points:

1 . Week 0 (full-dose treatment vitamin A supplement commenced);

2. Week 3;

3. Week 7 (maintenance dose treatment with vitamin A supplement);

4. Week 8 (Reinjury, full-dose treatment with vitamin A supplement restored);

5. Week 11 (PRP administered, whilst full-dose treatment with vitamin A supplement continued);

6. Week 20 (full work commenced);

7. Week 24.

Ultrasonography images were captured of the injured tendon. To determine the lesion size as a percentage of the entire cross-sectional area of the tendon, the lesion in the ultrasonogram image was measured manually, aided by online software. The echogenicity of the injured tendon was assessed by comparing grey scale statistics of the tendon with the values of a healthy adjacent tendon tissue. Due to equine anatomy, there is always a directly adjacent healthy tendon to use as a control (ultrasonography is not very affected by depth of tissue). The statistics comprised determining the mean pixel intensity at a number of random points on the injured tendon and the healthy tendon, and calculating a mean value for the echogenicity of each tissue. A ratio of mean echnogenicity of the injured tendon and the healthy tissue was determined to assess the extent of damage/healing at the lesion.

Results: The results are shown in Figure 1. Lesion size (left hand axis) is represented in green. Echogenicity values (right hand axis) are shown in blue and red. The blue line shows a plot of the values for the ratio of lesion: healthy tissue. This provides an assessment of the echogenicity of the main lesion. The red line shows a plot of the values for the ratio of injured: healthy tissue. This provides an assessment of the echogenicity of the rest of the injured tendon.

The plots in Figure 1 show that, over the first 7 weeks of full-dose treatment with vitamin A supplement, the lesion size decreased dramatically from just under 20% to -2.5%. The values for the echogenicity ratios also increased over this period, achieving near full resolution of her injury. Following reinjury (week 8), the lesion size increased to 21.52% of the tendon, and the values for the echogenicity ratios fell to -0.350 and -0.750 for the blue and red lines, respectively. At week 11 (3 weeks after restarting full-dose treatment), the lesion size was similar, at 20.62%, but with considerable improvements in echogenicity values (-0.900 and -1.050 for the blue and red lines, respectively).

By week 20 (-9 weeks after administration of PRP), the lesion size had shrunk considerably, to 2.01% of the entire tendon, although the values for the echogenicity ratios appeared to have slightly deteriorated (-0.700 and -0.900 for the blue and red lines, respectively), but still showed substantial improvement compared to baseline. Rania was able to start full work again from week 20.

The scan at week 24 shows that the lesion size measures 2.19%, which is stable from the scan at week 20 (showing 2.01%). The echogenicity of the main lesion improved (demonstrated by the blue line in Figure 1) and the echogenicity of the rest of the injured tendon remained stable (see the red line in Figure 1). By this stage, the benefits from the combination therapy may largely be plateauing, although there may still be some remodelling of the main scar tissue. The values which are plateauing are almost identical to the values achieved at week 7 (just before the re-injury).

Conclusions:

It was concluded from these results that full-dose treatment with vitamin A supplement resulted in substantial healing of the original tendon injury. Despite a nasty reinjury, combination treatment with PRP and vitamin A supplement resulted in improvement up to the same point as before, even though a reinjury would not be expected to recover as well.

These results appear to support an interpretation that administration of PRP resets the injury healing process, as shown by the apparent ‘worsening’ echogenicity by week 20 accompanied by substantial reduction in lesion size (-20% to -2%). An alternative explanation for the mild worsening of echogenicity is that tissue regeneration was hindered due to the lack of physical stimulus as Rania was box rested.

The synergy between the vitamin A and PRP combination treatment seems to facilitate the lesion to achieve continued regeneration of the injured tissue more so than just vitamin A or PRP alone.

Example 2

Effect of post-treatment maintenance dose of vitamin A supplement on connective tissue injuries

This example shows the effect of administering a post-treatment maintenance dose of vitamin A supplement on treating connective tissue injuries in horses. The results provide evidence for continuing at least a maintenance dose of vitamin A supplement past 7 weeks for connective tissue injury healing progression.

Methodology:

Full length ultrasonography of the injured tendon/ligament were performed at baseline as well as 3 weeks, 5 weeks and 7 weeks into the trial. Ultrasonography images were captured at the site of maximal injury. Depending on the nature of the injury, either cross-sectional or longitudinal views were taken. The lesions in the cross-sectional images were measured manually, aided by online software to determine the lesion size as a percentage of the overall cross-sectional area. The longitudinal images were presented to a consultant musculoskeletal radiologist who applied a 5-level grading system of the appearance of the injury corresponding to approximately 0%, 25%, 50%, 75% and 100% lesion size. Side effects and tolerability were also recorded.

Vitamin A supplement used:

Maintenance dose of vitamin A palmitate (retinyl palmitate) in a vehicle, delivered in dry feed at 8,000 IU per kg feed dry matter.

Subjects:

14 horses with 15 injured limbs took part in the study. The injuries comprised 9 tendon and 6 ligament injuries, with 12 injuries on the left side of the horse and 3 on the right. The mean time since injury was ~12 months. The tendon injuries comprised 2 acute injuries (<1 month old) and 7 chronic injuries, with a mean time since injury of 13.1 months, and a range of 9- 20 months. The ligamentous injuries comprised 6 chronic injuries, with a mean time since injury of 14.3 months and a range of 4-30 months. The most commonly injured structure was the left fore superficial digital flexor tendon (n = 8). The most commonly injured ligament was the left fore check ligament. The inclusion criteria were polo horses with diagnosed tendon/ligament injuries regardless of time since injury. There were no exclusion criteria.

Administration of vitamin A supplement:

At week 7 of full-treatment with vitamin A supplement, 7 horses with connective tissue injuries were orally administered vitamin A palmitate in a vehicle, at a dose of 80,000 III vitamin A once per day for 7 weeks (half the treatment dose of vitamin A supplement). The known toxic dose in horses is 1 ,000 IU per kg (National Research Council. Nutrient Requirement of Horses: Fifth Revised Edition. The National Academies. 1989)). The dose of 80,000 IU corresponds to 16% of the toxic dose, assuming a 500kg horse consuming 10kg of dry feed. There were no adverse events reported and the supplement was well tolerated by the subjects. The remaining 7 horses were orally administered a placebo in a vehicle, delivered in dry feed. The trial was a double-blind randomised controlled trial.

Results

The effect of post-treatment maintenance dose of vitamin A supplement on lesion size was investigated. Figure 2(a) shows the mean lesion size from week 0 to week 7 on full-treatment doses of vitamin A supplement before splitting into the values of the maintenance dose and placebo groups. The calculated percentage improvement from baseline to week 7 was deemed to be normally distributed and paired t-tests showed that the decrease from baseline to weeks 5 and 7 were statistically significant (p = <0.001 and 0.006 respectively). From week 7 to week 14, the mean lesion size of the placebo group increased from 31 .73% to -35.50%. The mean lesion size of the maintenance dose group decreased from 31.73% to -20.00% from week 7 to week 14. The data shows that there was continued improvement in the healing of injured connective tissues for subjects that were administered maintenance dose of vitamin A supplement, and deterioration of the lesions for subjects that were administered placebo.

It was possible that the above results may give a slightly biased impression of the effect of the maintenance dose as the subjects in both groups (maintenance vs placebo) had different intragroup mean lesion sizes at week 7. Figure 2(b) addresses this issue by showing the progression of both groups as well as the average value from Week 0 through to Week 14. Both groups continued to improve from week 7, however the figure still shows a more dramatic improvement in subjects taking a maintenance dose compared to those on placebo (-24.5% vs -6.28%).

One subject became re-injured after treatment with full-treatment doses of vitamin A supplement stopped and a maintenance dose of vitamin A supplement was administered for 7 weeks. Figure 3 shows an ultrasonogram at baseline (Fig. 3(a)), week 7 (7 weeks of treatment with full-treatment doses of vitamin A supplement) (Fig. 3(b)), and week 14 (7 weeks of treatment with maintenance dose vitamin A supplement) (Fig. 3(c)). The size of the lesion decreases at 7 weeks of full-treatment doses, as shown by a reduction in the hypoechoic area of the ultrasonogram (Figure 3(b)). However, the lesion becomes much more hypoechoic after post-treatment maintenance dose has been administered for 7 weeks (Figure 3(c)). Thus, the health of this particular lesion deteriorated after treatment with maintenance dose of vitamin A supplement. This result is different to those described above, wherein the maintenance dose cohort showed improvement in the regeneration of the lesion. The re-injury presented in Figure 3 provides evidence that, in some cases, treatment with full-treatment doses of vitamin A may be required for longer periods for regeneration of the tissue.

Conclusion

It is to be decided on a case-by-case basis whether maintenance dose is suitable for the subject. Some subjects will need to remain on full-dose treatment until optimum regeneration of the connective tissue has occurred to minimise the chances of re-injury occurring.

Example 3

Effect of treatment and activity level on connective tissue improvement

This example shows the effect of activity level on connective tissue improvement in horses on vitamin A supplement.

The subjects of the investigation are the same as those of Example 2 above.

Methodology

Improvement (as a percentage of baseline) of the injured tendon/ligament was performed at 7 weeks into the trial. All 14 horses were administered full-treatment doses of vitamin A supplement (vitamin A palmitate (retinyl palmitate) in a vehicle, delivered in dry feed at 16,000 III per kg feed dry matter) from week 0 to week 7 whilst being put to work. 7 horses were put on light/no work and 7 on full work. The allocation of each horse into one of the two groups was determined by the respective owner of the horse, who decided based on whether they could afford for the horse to be put on light work (and thus investigate the isolated effect of vitamin A supplement on lesion progression) or whether the horse was required to be put back to full work.

Results

Figure 4 shows the effect of activity level on the healing of connective tissue injury. The graph shows that the horses put on light or no work (n=7) demonstrated less of an improvement in the health of the tendon/lesion (30.35% improvement) than the horses put on full work (45.05% improvement) (n=7).

These results suggest that the effect of vitamin A supplement on connective tissue injury is enhanced by increasing activity. However, increasing activity too much is likely to result in a decrease in percentage improvement and worsening of the lesion (not shown in this example).

Example 4 - Treatment of equine connective tissue injuries

This example describes the effect of a vitamin A supplement in treating connective tissue injuries in horses.

Connective tissue injuries are common in both human and equine athletes with massive physical, psychological and economic impact. These tendon and ligament injuries tend to take weeks to months of recovery time mainly consisting of rest and rehabilitation, depending on the severity. There are various outcomes possible following injury. Commonly, disorganised scar tissue is formed to replace the native tissue to quickly restore form at the expense of future function. This scar tissue is characterised by a disorganised extracellular matrix which does not have the same mechanical properties and integrity of the original tendon. It follows logically that these athletes are never quite able to achieve optimum performance once scarring has occurred and are also prone to re-injury. It is traditionally believed that once a scar has formed, it is there for life as it is believed to be essentially a passively maintained disorganised cluster of collagen, even after remodelling. However, there is recent evidence that suggests that scar tissue is actively maintained which may present an avenue to target established fibrotic tissue (Fear M et al. Changes in Fibroblast Phenotype and Matrix Turnover in Established Scar Tissue, J Burn Care Res 2019, Vol 40, Page 237).

Vitamin A has multiple functions in animals involving (and not limited to) development, and modulation of protein synthesis, and also possesses anti-inflammatory properties. There is some evidence that vitamin A plays a role in scar tissue formation and maintenance. This example aims to demonstrate the safety, and establish the clinical efficacy, of the usage of vitamin A supplementation in horses with tendon or ligament injuries.

Methodology:

A prospective, single armed pilot trial of the efficacy and safety of Vitamin A supplementation in connective tissue injuries in horses was performed. The time since injury of each horse was noted. Full length ultrasonography of the injured tendon/ligament were performed at baseline as well as 3 weeks, 5 weeks and 7 weeks into the trial. Ultrasonography images were captured at the site of maximal injury. Depending on the nature of the injury, either cross-sectional or longitudinal views were taken. Figure 5(a) shows a cross-sectional ultrasonogram of the major tendons/ligaments present in the left foot of a horse (normal equine anatomy). Figure 5(b) shows a longitudinal ultrasonogram (left) of the left foot of a horse compared with a cross-sectional ultrasonogram (right) of the left foot of the same horse (normal equine anatomy). The lesions in the cross-sectional images were measured manually, aided by online software to determine the lesion size as a percentage of the overall cross-sectional area. The longitudinal images were presented to a consultant musculoskeletal radiologist who applied a 5-level grading system of the appearance of the injury corresponding to approximately 0%, 25%, 50%, 75% and 100% lesion size. Side effects and tolerability were also recorded.

Vitamin A supplement used:

Vitamin A palmitate (retinyl palmitate) in a vehicle, delivered in dry feed based on the upper safe concentration in feeds (16,000 IU per kg feed dry matter).

Administration of vitamin A supplement:

Horses with connective tissue injuries were orally administered vitamin A palmitate in a vehicle, at a dose of 160,000 IU vitamin A once per day for 7 weeks. The dose administered was decided based on the known toxic dose in horses (1 ,000 IU per kg (National Research Council. Nutrient Requirement of Horses: Fifth Revised Edition. The National Academies. 1989)) and the proposed upper safe concentration in feeds (16,000 IU per kg feed dry matter (Ralston SL. Nutritional Requirements of Horses and Other Equids. MSD Veterinary Manual, 2021)) which yielded a dose of 160,000 IU, corresponding to 32% of the toxic dose, assuming a 500kg horse consuming 10kg of dry feed. There were no adverse events reported and the supplement was well tolerated by the subjects.

Subjects:

14 horses with 15 injured limbs were enrolled in the study between March and May 2021. The injuries comprised 9 tendon and 6 ligament injuries, with 12 injuries on the left side of the horse and 3 on the right. The mean time since injury was ~12 months. The tendon injuries comprised 2 acute injuries (<1 month old) and 7 chronic injuries, with a mean time since injury of 13.1 months, and a range of 9-20 months. The ligamentous injuries comprised 6 chronic injuries, with a mean time since injury of 14.3 months and a range of 4-30 months. The most commonly injured structure was the left fore superficial digital flexor tendon (n = 8). The most commonly injured ligament was the left fore check ligament. The inclusion criteria were polo horses with diagnosed tendon/ligament injuries regardless of time since injury. There were no exclusion criteria.

Statistical analysis:

All statistical analysis was performed on SPSS v27. Missing values were handled by last observation carried forward. Descriptive statistics were used to describe baseline and followup values. The results were first tested for normality using the Shapiro-Wilk test. The results at weeks 3, 5 and 7 were then analysed compared to baseline using two-tailed paired Student’s t-tests once normality was proven. One-tailed Pearson correlation test was performed to determine if outcomes was correlated with time since injury as we hypothesise that older, more established injuries may benefit less from our supplementation.

Results:

The mean lesion size was 41.44% at baseline, 35.87% at week 3, 28.37% at week 5 and 31 .73% at week 7 (Figure 6(a)). Shapiro-Wilk tests on the data revealed the lesion size was normally distributed at baseline and at weeks 3, 5 and 7. The calculated percentage improvement from baseline to week 7 was also deemed to be normally distributed. Thereafter, paired t-tests showed that the decrease from baseline to weeks 5 and 7 were statistically significant (p = <0.001 and 0.006 respectively) (Figure 6(a)). When acute injury (<1 month) cases were removed (n = 2), similar results were obtained with statistical significance being achieved at weeks 5 and 7 compared to baseline (p = <0.001 and p = 0.017 respectively). Figures 7 to 18 show ultrasonograms of some of the tendon/ligamentous injuries of the data set at various time points from week 0. Table 1 shows the results of the trial, comprising data for each individual lesion.

One-tailed Pearson correlation coefficient on percentage improvement and time yielded a test statistic of r = -0.508 (p = 0.027) indicating a statistically significant negative relationship (Figure 6(b)). However, removal of 3 outliers yielded r = -0.806 (p = 0.003).

Table 1. Full results table.

Note: LF - left fore, RF - right fore, LH - left hind, RH - right hind, SDFT - superficial digital flexor tendon

Discussion:

The results of the study show that supplementation with vitamin A was extremely well tolerated with no signs of vitamin A toxicity noted in all subjects. Owing to the fact that the subjects were all specialised polo horses, there was a higher incidence of left sided injuries.

Our results show that it takes approximately 5 weeks for there to be significant improvements in the appearance of tendon/ligament injuries in horses which is maintained at least up to week 7 (Figure 6(a)). Given that more than half of the injuries occurred over 12 months prior to the study and thus were very likely to be stable at the start of the trial, a noticeable improvement in 5 weeks is very astonishing. The results also show that there is a statistically significant linear negative correlation between the time between injury and starting vitamin A supplementation and outcomes (Figure 6(b)), indicating that earlier vitamin A supplementation leads to better outcomes.

The dataset had three outliers as shown in scatter plot in Figure 6(b): two at 12 months since injury, which both had 100% improvement, and one at 20 months since injury which had - 25% improvement (i.e. the lesion got worse). The explanation for the outlier at 20 months since injury was that the ligamentous injury sustained by the subject was confirmed by a consultant radiologist to be a complete avulsion of the ligament. Short of surgery, there is no possibility of the ligament recovering following that type of injury, including with vitamin A supplement. The two outliers at 12 months since injury both had their lesions become unnoticeable by 7 weeks which is extremely promising.

General wound healing has 4 overlapping stages - haemostasis, inflammation, tissue formation and remodelling. Recent advancements in the field of wound healing suggest that the lack of inflammation in foetal wounds allows it to heal in a scarless manner, restoring the full function, flexibility and architecture of the native tissue (Galatz LM et al. Tendon Regeneration and Scar Formation: The Concept of Scarless Healing. J Orthop Res. 2015; 33:823-31). The known anti-inflammatory properties of vitamin A (Huang Z et al. Role of Vitamin A in the Immune System. J Clin Med. 2018;7(9):258) support its use in the acute stages of tendon injuries, and indeed has been shown to increase the tensile strength of the healed tendon by double the control at day 45 in a 1990 study on chickens in Greenwald et al. Zone II Flexor Tendon Repair: Effects of Vitamins A, E, /3-carotene. J Surg Res. 1990;49(1):98-102, but does not explain the improvement found in the subjects of our study that have long-standing injuries. The healing of tendon injuries starts with an early deposition of unoriented collagen fibres. Later on, a dynamic interplay of collagenolysis and deposition of oriented fibres determine the extent of restoration of normal tissue architecture (Greenwald et al., supra). Traditional thinking is that this process plateaus and leaves a permanent fibrotic scar. From our study, the fact that long-standing injuries (>12 months old) showed signs of improvement on ultrasonography is very encouraging especially with the recent suggestion that scar tissue is actively maintained. Fear et al. (supra) suggested this by demonstrating that fibroblasts in scar tissue are phenotypically different to fibroblasts in normal skin which is linked to the difference in matrix turnover.

The main cell type present in tendons are tenocytes (also known as tendon fibroblasts) and these maintain the tendon extracellular matrix ECM. Tendons are characterised by an exceptionally organised, anisotropic extracellular matrix with primarily type I collagen, although small amounts of type III collagen are also present (Fratzl P. Collagen: Structure and Mechanics, an Introduction. Collagen. Springer US; 2008. p. 1-13; Kannus P. Structure of the Tendon Connective Tissue. Scand J Med Sci Sport. 2000; 10(6): 312-20). Equine tendon scar tissue has been shown to have higher than usual levels of type III collagen (20- 30%) (Williams I et al. Cell Morphology and Collagen Types in Equine Tendon Scar. Res Vet Sci. 1980;28:302-10). Vitamin A is well known to play a role in the modulation of the synthesis of extracellular matrix proteins, including collagens, laminins, entactin, fibronectin, elastin and proteoglycans. It also has a role in the expression of various metalloproteinases, including collagenase. As scar tissue is due to excess deposition of disoriented collagen and physiologically abnormal proportions of collagen type by fibroblasts, this may allude to a plausible mechanism how vitamin A may influence fibroblasts maintaining scar tissue to instead produce native tendon tissue.

Conclusion

In summary, our study found that vitamin A supplementation in horses with established tendon and ligament injuries led to radiological improvement and thus may positively influence the actively maintained characteristic extracellular matrix of scar tissue. We also found evidence that the degree of improvement with this supplementation is correlated with the time since injury. A potential mechanism by which it may act is upon fibroblasts or possibly their progenitor cells, mesenchymal stem cells. As scarring and fibrosis are seen in almost all areas of medicine, these findings have substantial implications and potential therapeutic uses if the mechanistic pathways are found to be more widely applicable. Example 5 - Treatment of equine connective tissue injuries

This example describes the pathophysiology of equine tendon/ligament lesions before and after treatment with vitamin A supplement.

Methodology:

Full length ultrasonography of the injured tendon/ligament was performed at the time of initial injury, baseline, as well as 3 weeks, 5 weeks and 7 weeks into the trial. Ultrasonography images were captured at the site of maximal injury. Depending on the nature of the injury, either cross-sectional or longitudinal views were taken. The lesions in these cross-sectional images were measured manually, aided by online software to determine the lesion size as a percentage of the overall cross-sectional area. The longitudinal images were presented to a consultant musculoskeletal radiologist who applied a 5-level grading system of the appearance of the injury corresponding to approximately 0%, 25%, 50%, 75% and 100% lesion size. Side effects and tolerability were also recorded.

The administration of vitamin A supplement, and the vitamin A supplement used, were the same as that of Example 4.

Subjects:

7 horses with 7 injured limbs were enrolled in the study between March and May 2021. The injuries comprised 3 tendon and 4 ligament injuries. The inclusion criteria were polo horses with diagnosed tendon/ligament injuries regardless of time since injury. The exclusion criteria were horses with acute connective tissue injuries.

Statistical analysis:

All statistical analysis was performed on SPSS v27. Missing values were handled by last observation carried forward. Descriptive statistics were used to describe baseline and followup values. The results were first tested for normality using the Shapiro-Wilk test. The results at weeks 3, 5 and 7 were then analysed compared to baseline using two-tailed paired Student’s t-tests once normality was proven.

Results

Figure 19(a) shows the size of the 7 lesions under investigation from the time of original injury, at baseline, and at 3, 5, and 7 weeks into the trial. The mean lesion size was 43.97% at the time of original injury and 49.43% at baseline (time treatment with vitamin A supplement began), but this difference was not statistically significant. However, when tendon and ligament lesions were treated as two separate cohorts, there was an apparent divergence in natural healing progression between the two tissues from the time of original injury to when treatment began. As shown in Figure 19(b), the tendons (n=3) tended to get worse as shown by an increase in lesion size across all three tendons, which was statistically significant (mean increase in lesion size = 48.7%, p = 0.03). Confirming the findings from previous analyses (Example 4), there was a statistically significant improvement in lesion size from baseline to week 7 with treatment with vitamin A supplement (mean improvement in lesion size = 30.62% p = 0.04).

As shown in Figure 19(c), the ligaments tended to get better, demonstrated by a reduction in lesion size across three out of four lesions from the time of original injury to the start of treatment. The lesion that increased in size was an extremely severe injury, and the only subject across the entire study that did not improve even after treatment with vitamin A supplement. When analysing all 4 ligamentous injuries, there was no significant difference in lesion size at the time of original injury and the start of the trial (Figure 19(c)). However, when excluding the known outlier, it was found that there was a statistically significant reduction in lesion size between the time of original injury and the start of the trial (Figure 19(d)). There was also a reduction in the size of two of the three lesions of the data set from baseline to week 7 with treatment with vitamin A supplement (Figure 19(d)). The lesion that stayed the same size from week 0 to week 7 was the oldest injury in the trial (30 months since original injury).

Discussion

Interestingly, it appears that tendons and ligaments behave differently over their natural healing processes with ligamentous lesions either getting better or roughly staying the same from the time of original injury before improving with vitamin A supplement, and tendonous injuries getting significantly worse before improving with vitamin A supplement. These data suggest that there is a physiological difference between tendons and ligaments that results in diverging progression through wound healing, which provides guidance for the next steps. Example 6 - Treatment of equine tendon injury

This example shows the effect of vitamin A supplement on the echogenicity and health of tendon injury.

Structures composed of different tissue will have different echogenicities. The health of a tissue such as a tendon can be assessed by comparing the echogenicity of the tissue with that of a corresponding healthy tissue. Healthy tendons comprising native tissue and normal architecture are usually hyperechoic and appear white on the sonogram; they are capable of reflecting ultrasound that is cast over the tissue. An injured tendon comprising a lesion will appear as less hyperechoic, and more hypoechoic as tendon fibers are interrupted and defects are usually filled with fluid, blood, or fat. Severe lesions will be anechoic, and will display as completely dark on the sonogram.

Methodology:

Full length ultrasonography of the injured tendon and adjacent healthy tendon was performed at baseline as well as 3 weeks, 5 weeks and 7 weeks into the trial. Ultrasonography images were captured at the site of maximal injury of the injured tendon. Depending on the nature of the injury, either cross-sectional or longitudinal views were taken. The echogenicity of a tendon lesion was assessed by comparing grey scale statistics of the tendon lesion with the values of healthy adjacent tendon tissue (Figure 20(b)). Due to equine anatomy, there is always a directly adjacent healthy tendon to use as a control (ultrasonography is not very affected by depth of tissue). The statistics comprised determining the mean pixel intensity at a number of random points on the lesion and the healthy tendon, and calculating a mean value for the echogenicity of each tissue. A ratio of mean echnogenicity of the lesion and the healthy tissue was determined to assess the extent of damage/healing at the lesion.

Vitamin A supplement used, its administration, and the subjects investigated, comprise those of Example 4.

Results

The mean echogenicity ratio of the lesion and healthy tendon increased each week from baseline to week 7 (Figure 20(a)), which was statistically significant. A value of 1 would indicate perfect regeneration of native tendon. The mean echogenicity ratio was 0.52 at baseline, and 0.69 at week 7, equating to a % increase of 32.7%. As shown in Figure 20(a), the mean echogenicity ratio from baseline to week 7 showed a continuing positive gradient. This shows that the lesion is being replaced with native tendon tissue at a constant rate, providing evidence for continuing trials with full dose of vitamin A supplement past 7 weeks to achieve even more regeneration of the tendon. Figure 20(b) shows an outline of the whole injured tendon and area of lesion. Figure 20(c) shows an outline of an adjacent healthy tendon (as well as the outline of the injured tissue and lesion) used as a comparison tissue.

Interestingly, the injured tendon had lower echogenicity than the healthy tendon when the area of lesion was excluded and the remaining area of the injured tendon was examined (Figure 21(b). Figure 21(a) shows that the mean echogenicity ratio at week 0 was 0.76, which increased marginally to 0.82 at week 7, which was statistically significant. It was observed that the lesion size had normalised to the size of the tendon itself, suggesting that the lesion was not contained to the point of maximal injury, but that the health of the whole tendon was impaired.

Claims

45 Claims

1 . A method of treating a connective tissue injury in a subject comprising administering to the subject an effective amount of vitamin A and platelet-rich plasma (PRP).

2. A method according to claim 1 , wherein the connective tissue injury is a tendon injury.

3. A method according to claim 1 , wherein the connective tissue injury is a ligament injury.

4. A method according to any preceding claim, wherein the vitamin A and the PRP are administered sequentially to the subject.

5. A method according to claim 4, wherein the vitamin A is administered before the PRP.

6. A method according to any preceding claim, wherein a plurality of doses of the vitamin A is administered to the subject prior to administration of PRP.

7. A method according to any of claims 1 to 3, wherein the vitamin A and the PRP are co-administered to the subject.

8. A method according to any preceding claim, wherein the vitamin A is administered to the subject at least once per day.

9. A method according to any preceding claim, wherein the vitamin A is administered to the subject at least once per day for at least 3 days from the day of first administration to the subject.

10. A method according to any preceding claim, wherein the vitamin A is administered to the subject at least once per day for at least a week, at least a month, or at least 6 months from the day of first administration to the subject.

11. A method according to any preceding claim wherein the vitamin A is first administered to the subject within a week of the injury.

12. A method according to any preceding claim, wherein the PRP is first administered to the subject at least three weeks after first administration of vitamin A.

13. A method according to any preceding claim, wherein the PRP is administered once a ratio of echogenicity of injured connective tissue of the connective tissue injury to echogenicity of corresponding uninjured connective tissue is greater than 0.8.

14. A method according to any preceding claim, wherein the vitamin A comprises isolated vitamin A. 46

A method according to any preceding claim, wherein the vitamin A comprises a preformed vitamin A, such as a retinyl ester or retinol.

16. A method according to any preceding claim, wherein the vitamin A comprises a provitamin A, such as a carotenoid.

17. A method according to any preceding claim, wherein the vitamin A comprises a bioactive form of vitamin A, such as retinal or retinoic acid.

18. A method according to any preceding claim, wherein the vitamin A is administered to the subject at a dose which comprises up to 50% of a maximum safe dose of vitamin A for the subject per day, or >10% to 50%, or 25% to 50% of a maximum safe dose of vitamin A for the subject per day.

19. A method according to any preceding claim, wherein the vitamin A is administered at a dose which comprises up to 50% of a minimum toxic dose for the subject.

20. A method according to any preceding claim, wherein the vitamin A is administered at a dose which comprises at least 5% of a minimum toxic dose for the subject.

21 . A method according to any preceding claim, wherein the subject is a human subject.

22. A method according to claim 21 , wherein the vitamin A is administered to the subject at a dose of >10,000 to 100,000 III vitamin A per day.

23. A method according to claim 22, wherein the vitamin A is administered to the subject at a dose of about 25,000-100,000, 50,000-100,000, or 75,000-100,000 IU vitamin A per day.

24. A method according to any of claims 1 to 20, wherein the subject is a horse.

25. A method according to claim 24, wherein the horse is administered a dose of 25,000- 250,000, 50,000-250,000, 75,000-250,000, 100,000-250,000, 125,000-250,000, 150,000- 250,000, 175,000-250,000, or 200,000-250,000 IU vitamin A per day.

26. A method according to claim 25, wherein the horse is administered a dose of 25,000- 50,000, 25,000-75,000, 25,000-100,000, 25,000-125,000, 25,000-150,000, 25,000-175,000, or 25,000-200,000 IU vitamin A per day.

27. A method according to any preceding claim wherein the vitamin A is administered systemically to the subject.

28. A method according to claim 27 wherein the vitamin A is administered orally or intravenously to the subject. 47

29. A method according to any preceding claim, wherein the PRP is administered to the subject at a dose wherein the platelet concentration is at least 1 ,000,000 platelets/pl.

30. A method according to any preceding claim wherein the PRP is administered to the subject at least once every month, or once every six months.

31. A method according to any preceding claim wherein the PRP is administered intravenously to the subject.

32. A method according to any preceding claim wherein the PRP comprises platelets obtained from the subject.

33. A method according to any preceding claim wherein the PRP is autologous PRP.

34. A method according to claim 33, wherein the PRP comprises additional growth factors.

35. A method according to any preceding claim, wherein the subject is administered a maintenance dose of vitamin A, wherein the maintenance dose is less than a full treatment dose.

36. A method according to claim 35, wherein the maintenance dose is up to three quarters of a full treatment dose.

37. A method according to claim 35 or 36, wherein the maintenance dose is up to two- thirds of a full treatment dose.

38. A method according to any of claims 35 to 37, wherein the maintenance dose is at least a quarter of a full treatment dose.

39. A method according to any of claims 35 to 38, wherein the maintenance dose is administered after the subject has been administered one or more full-treatment doses.

40. A method according to any of claims 35 to 39, wherein the maintenance dose is administered from the day after the last administration of a full-treatment dose to the subject.

41. A method according to any of claims 35 to 40, wherein a plurality of maintenance doses is administered to the subject.

42. A method according to claim 41 , wherein the maintenance doses are administered for at least one week from the day of first administration of a maintenance dose to the subject.

43. A method according to claim 42, wherein the maintenance doses are administered for at least 4 weeks from the day of first administration of a maintenance dose to the subject.

44. A method according to any of claims 35 to 43, wherein the subject is administered PRP at least one week after the last administration of a maintenance dose of vitamin A to the subject.

45. A method according to any of claims to 35 to 44, wherein the subject is a human subject.

46. A method according to claim 45, wherein the, or each maintenance dose comprises >2,500 III to 75,000 IU vitamin A per day.

47. A method according to claim 46, wherein the, or each maintenance dose comprises 5,000-75,000, 10,000-75,000, or 20,000-75,000 IU vitamin A per day.

48. A method according to claim 47, wherein the, or each maintenance dose comprises 5,000-50,000, 10,000-50,000, or 20,000-50,000 IU vitamin A per day.

49. A method according to any of claims 35 to 44, wherein the subject is a non-human subject.

50. A method according to claim 49, wherein the subject is a horse.

51. A method according to claim 50, wherein the, or each maintenance dose comprises 10,000-200,000, 20,000-150,000, 40,000-100,000, or 60,000-100,000 IU vitamin A per day.

52. Vitamin A and PRP, for use in the treatment of a connective tissue injury in a subject.

53. PRP for use in the treatment of a connective tissue injury in a subject administered Vitamin A.

54. Vitamin A for use in the treatment of a connective tissue injury in a subject administered PRP.

55. Vitamin A and PRP for use according to claim 52, PRP for use according to claim 53, or vitamin A for use according to claim 54, wherein the connective tissue injury is a tendon injury.

56. Vitamin A and PRP for use according to claim 52, PRP for use according to claim 53, or vitamin A for use according to claim 54, wherein the connective tissue injury is a ligament injury.

57. Vitamin A and PRP for use according to any of claims 52, 55, or 56, wherein the vitamin A and PRP are for sequential administration to the subject.

58. Vitamin A and PRP for use according to claim 57, wherein the vitamin A is for administration to the subject prior to the PRP.

59. Vitamin A and PRP for use according to claim 58, wherein a plurality of doses of vitamin A is to be administered to the subject prior to administration of PRP.

60. Vitamin A and PRP for use according to any of claims 52, 55 or 56, wherein the vitamin A and PRP are for co-administration to the subject.

61. Vitamin A and PRP for use according to any of claims 52, or 55 to 60, or vitamin A for use according to claim 54, wherein the vitamin A is to be administered to the subject at least once per day.

62. Vitamin A and PRP for use according to any of claims 52, or 55 to 61 , or vitamin A for use according to claim 54 or 61 , wherein the vitamin A is to be administered to the subject at least once per day for at least 3 days from the day of first administration to the subject.

63. Vitamin A and PRP for use according to any of claims 52, or 55 to 62, or vitamin A for use according to claim 54, 61 , or 62, wherein the vitamin A is to be administered to the subject at least once per day for at least a week, at least a month, or at least 6 months from the day of first administration to the subject.

64. Vitamin A and PRP for use according to any of claims 52, or 55 to 63, or vitamin A for use according to any of claims 54, or 61 to 63, wherein the vitamin A is first to be administered to the subject within a week of the injury.

65. Vitamin A and PRP for use according to any of claims 52, or 55 to 64, or PRP for use according to claim 52, wherein the PRP is first to be administered to the subject at least three weeks after first administration of vitamin A.

66. Vitamin A and PRP for use according to any of claims 52, or 55 to 65, or PRP for use according to claim 52 or 65, wherein the PRP is to be administered to the subject once a ratio of echogenicity of injured connective tissue of the connective tissue injury to echogenicity of corresponding uninjured connective tissue is greater than 0.8.

67. Vitamin A and PRP for use according to any of claims 52, or 55 to 66, vitamin A for use according to any of claims 54, or 61 to 63, or PRP for use according to any of claims 52, 65, or 66, wherein the vitamin A comprises isolated vitamin A.

68. Vitamin A and PRP for use according to any of claims 52, or 55 to 67, vitamin A for use according to any of claims 54, 61 to 63, or 67, or PRP for use according to any of claims 52, or 65 to 67, wherein the vitamin A comprises a preformed vitamin A, such as a retinyl ester or retinol.

69. Vitamin A and PRP for use according to any of claims 52, or 55 to 68, vitamin A for use according to any of claims 54, 61 to 63, 67, or 68, or PRP for use according to any of claims 52, or 65 to 68, wherein the vitamin A comprises a provitamin A, such as a carotenoid.

70. Vitamin A and PRP for use according to any of claims 52, or 55 to 69, vitamin A for use according to any of claims 54, 61 to 63, or 67 to 69, or PRP for use according to any of claims 52, or 65 to 69, wherein the vitamin A comprises a bioactive form of vitamin A, such as retinal or retinoic acid.

71. Vitamin A and PRP for use according to any of claims 52, or 55 to 70, or vitamin A for use according to any of claims 54, 61 to 63, or 67 to 70, wherein the vitamin A is to be administered to the subject at a dose which comprises up to 50% of a maximum safe dose of vitamin A for the subject per day, or >10% to 50%, or 25% to 50% of a maximum safe dose of vitamin A for the subject per day.

72. Vitamin A and PRP for use according to any of claims 52, or 55 to 71 , or vitamin A for use according to any of claims 54, 61 to 63, or 67 to 71 , wherein the vitamin A is to be administered at a dose which comprises up to 50% of a minimum toxic dose for the subject.

73. Vitamin A and PRP for use according to any of claims 52, or 55 to 72, or vitamin A for use according to any of claims 54, 61 to 63, or 67 to 72, wherein the vitamin A is to be administered at a dose which comprises at least 5% of a minimum toxic dose for the subject.

74. Vitamin A and PRP for use according to any of claims 52, or 55 to 73, vitamin A for use according to any of claims 54, 61 to 63, or 67 to 73, or PRP for use according to any of claims 52, or 65 to 69, wherein the subject is a human subject.

75. Vitamin A and PRP for use according to claim 74, vitamin A for use according to claim

74, or PRP for use according to claim 74, wherein the vitamin A is to be administered to the subject at a dose at a dose of >10,000 to 100,000 III vitamin A per day.

76. Vitamin A and PRP for use according to claim 75, vitamin A for use according to claim

75, or PRP for use according to claim 75, wherein the vitamin A is to be administered to the subject at a dose of about 25,000-100,000, 50,000-100,000, or 75,000-100,000 IU vitamin A per day. 51

77. Vitamin A and PRP for use according to any of claims 52, or 55 to 73, vitamin A for use according to any of claims 54, 61 to 63, or 67 to 73, or PRP for use according to any of claims 52, or 65 to 69, wherein the subject is a horse.

78. Vitamin A and PRP for use according to claim 77, vitamin A for use according to claim 77, or PRP for use according to claim 77, wherein the vitamin A is to be administered to the horse at a dose of 25,000-250,000, 50,000-250,000, 75,000-250,000, 100,000-250,000, 125,000-250,000, 150,000-250,000, 175,000-250,000, or 200,000-25 250,000 IU vitamin A per day.

79. Vitamin A and PRP for use according to claim 77, vitamin A for use according to claim 77, or PRP for use according to claim 77, wherein the vitamin A is for administration to the horse at a dose of 25,000-50,000, 25,000-75,000, 25,000-100,000, 25,000-125,000, 25,000- 150,000, 25,000-175,000, or 25,000-200,000 IU vitamin A per day.

80. Vitamin A and PRP for use according to any of claims 52, or 59 to 79, or vitamin A for use according to any of claims 54, 61 to 63, or 67 to 79, wherein the vitamin A is for systemic administration to the subject.

81. Vitamin A and PRP for use according to claim 80, or vitamin A for use according to claim 80, wherein the vitamin A is for oral or intravenous administration to the subject.

82. Vitamin A and PRP for use according to any of claims 52, or 55 to 81 , or PRP for use according to any of claims 52, or 65 to 79, wherein the PRP is to be administered to the subject at a dose wherein the platelet concentration is at least 1 ,000,000 platelets/pl.

83. Vitamin A and PRP for use according to any of claims 52, or 55 to 82, or PRP for use according to any of claims 52, 65 to 79, or 82, wherein the PRP is to be administered to the subject once every month, or once every six months.

84. Vitamin A and PRP for use according to any of claims 52, or 55 to 83, or PRP for use according to any of claims 52, 65 to 79, 82 or 83, wherein the PRP is for intravenous administration to the subject.

85. Vitamin A and PRP for use according to any of claims 52, or 55 to 84, or PRP for use according to any of claims 52, 65 to 79, or 82 to 84, wherein the PRP comprises platelets obtained from the subject.

86. Vitamin A and PRP for use according to any of claims 52, or 55 to 85, or PRP for use according to any of claims 52, 65 to 79, or 82 to 85, wherein the PRP is autologous PRP. 52

87 Vitamin A and PRP for use according to any of claims 52, or 55 to 86, or PRP for use according to any of claims 52, 65 to 79, or 82 to 86, wherein the PRP comprises additional growth factors.

88. Vitamin A and PRP for use according to any of claims 52, or 55 to 87, or vitamin A for use according to any of claims 54, 61 to 63, or 67 to 81, wherein the vitamin A is to be administered to the subject at a maintenance dose of vitamin A, wherein the maintenance dose is less than a full treatment dose.

89. Vitamin A and PRP for use according to claim 88, or vitamin A for use according to claim 88, wherein the maintenance dose is up to three quarters of a full treatment dose.

90. Vitamin A and PRP for use according to claim 88 or 89, or vitamin A for use according to claim 88 or 89, wherein the maintenance dose is up to two-thirds of a full treatment dose.

91. Vitamin A and PRP for use according to any of claims 88 to 90, or vitamin A for use according to any of claims 88 to 90, wherein the maintenance dose is at least a quarter of a full treatment dose.

92. Vitamin A and PRP for use according to any of claims 88 to 91, or vitamin A for use according to any of claims 88 to 91, wherein the maintenance dose is to be administered after the subject has been administered one or more full-treatment doses.

93. Vitamin A and PRP for use according to any of claims 88 to 92, or vitamin A for use according to any of claims 88 to 92, wherein the maintenance dose is to be administered from the day after the last administration of a full-treatment dose to the subject

94. Vitamin A and PRP for use according to any of claims 88 to 93, or vitamin A for use according to any of claims 88 to 93, wherein a plurality of maintenance doses is to be administered to the subject.

95. Vitamin A and PRP for use according to claim 94, or vitamin A for use according to claim 94, wherein the maintenance doses are to be administered for at least one week from the day of first administration of a maintenance dose to the subject.

96. Vitamin A and PRP for use according to claim 94, wherein the maintenance doses are to be administered for at least 4 weeks from the day of first administration of a maintenance dose to the subject.

97. Vitamin A and PRP for use according to any of claims 88 to 96, wherein the PRP is to be administered to the subject at least one week after the last administration of a maintenance dose of vitamin A to the subject. 53

98. Vitamin A and PRP for use according to any of claims 88 to 97, wherein the subject is a human subject.

99. Vitamin A and PRP for use according to claim 98, wherein the, or each maintenance dose comprises >2,500 III to 75,000 IU vitamin A per day.

100. Vitamin A and PRP for use according to claim 99, wherein the, or each maintenance dose comprises 5,000-75,000, 10,000-75,000, or 20,000-75,000 IU vitamin A per day.

101. Vitamin A and PRP for use according to claim 99, wherein the, or each maintenance dose comprises 5,000-50,000, 10,000-50,000, or 20,000-50,000 IU vitamin A per day.

102. Vitamin A and PRP for use according to any of claims 88 to 97, wherein the subject is a non-human subject.

103. Vitamin A and PRP for use according to claim 102, wherein the subject is a horse.

104. Vitamin A and PRP for use according to claim 103, wherein the, or each maintenance dose comprises 10,000-200,000, 20,000-150,000, 40,000-100,000, or 60,000-100,000 IU vitamin A per day.

105. Use of vitamin A and PRP in the manufacture of a medicament for the treatment of a connective tissue injury in a subject.

106. Use of PRP in the manufacture of a medicament for the treatment of a connective tissue injury in a subject administered vitamin A.

107. Use of vitamin A in the manufacture of a medicament for the treatment of a connective tissue injury in a subject administered PRP.

108. Use according to any of claims 105 to 107, wherein the connective tissue injury is a tendon injury.

109. Use according to any of claims 105 to 107, wherein the connective tissue injury is a ligament injury.

110. Use of vitamin A and PRP according to any of claims 105, 108 or 109, wherein the vitamin A and PRP are for sequential administration to the subject.

111. Use of vitamin A and PRP according to claim 110, wherein the vitamin A is for administration to the subject prior to the PRP.

112. Use according to claim 111 , wherein a plurality of doses of vitamin A is to be administered to the subject prior to administration of the PRP. 54

113. Use of vitamin A and PRP according to any of claims 105, 108 or 109, wherein the vitamin A and PRP are for co-administration to the subject.

114. Use according to any of claims 105, or 107 to 113, wherein the vitamin A is to be administered to the subject at least once per day.

115. Use according to any of claims 105, or 107 to 114, wherein the vitamin A is to be administered to the subject at least once per day for at least 3 days from the day of first administration to the subject.

116. Use according to any of claims 105, or 107 to 115, wherein the vitamin A is to be administered to the subject at least once per day for at least a week, at least a month, or at least 6 months from the day of first administration to the subject.

117. Use according to any of claims 105, or 107 to 116, wherein the vitamin A is first to be administered to the subject within a week of the injury.

118. Use according to any of claims 105, 106, or 108 to 117, wherein the PRP is first to be administered to the subject at least three weeks after first administration of vitamin A.

119. Use according to any of claims 105, 106, or 108 to 118, wherein the PRP is to be administered to the subject once a ratio of echogenicity of injured connective tissue of the connective tissue injury to echogenicity of corresponding uninjured connective tissue is greater than 0.8.

120. Use according to any of claims 105 to 119, wherein the vitamin A comprises isolated vitamin A.

Use according to any of claims 105 to 120, wherein the vitamin A comprises a preformed vitamin A, such as a retinyl ester or retinol.

122. Use according to any of claims 105 to 121 , wherein the vitamin A comprises a provitamin A, such as a carotenoid.

123. Use according to any of claims 105 to 122, wherein the vitamin A comprises a bioactive form of vitamin A, such as retinal or retinoic acid.

124. Use according to any of claims 105, or 107 to 123, wherein the vitamin A is to be administered to the subject at a dose which comprises up to 50% of a maximum safe dose of vitamin A for the subject per day, or >10% to 50%, or 25% to 50% of a maximum safe dose of vitamin A for the subject per day. 55

125. Use according to any of claims 105, or 107 to 124, wherein the vitamin A is for administration at a dose which comprises up to 50% of a minimum toxic dose for the subject.

126. Use according to any of claims 105, or 107 to 125, wherein the vitamin A is for administration at a dose which comprises at least 5% of a minimum toxic dose for the subject.

127. Use according to any of claims 105 to 126, wherein the subject is a human subject.

128. Use according to claim 127, wherein the vitamin A is to be administered to the subject at a dose at a dose of >10,000 to 100,000 IU vitamin A per day.

129. Use according to claim 128, wherein the vitamin A is to be administered to the subject at a dose of about 25,000-100,000, 50,000-100,000, or 75,000-100,000 IU vitamin A per day.

130. Use according to any of claims 105 to 126, wherein the subject is a horse.

131. Use according to claim 130, wherein the vitamin A is for administration to the horse at a dose of 25,000-250,000, 50,000-250,000, 75,000-250,000, 100,000-250,000, 125,000- 250,000, 150,000-250,000, 175,000-250,000, or 200,000-25 250,000 IU vitamin A per day.

132. Use according to claim 130, wherein the vitamin A is for administration to the horse at a dose of 25,000-50,000, 25,000-75,000, 25,000-100,000, 25,000-125,000, 25,000- 150,000, 25,000-175,000, or 25,000-200,000 IU vitamin A per day.

133. Use according to any of claims 105, or 107 to 132, wherein the vitamin A is for systemic administration to the subject.

134. Use according to any of claims 105, or 107 to 133, wherein the vitamin A is for oral or intravenous administration to the subject.

135. Use according to any of claims 105, 106, or 108 to 134, wherein the PRP is to be administered to the subject at a dose wherein the platelet concentration is at least 1 ,000,000 platelets/ l.

136. Use according to any of claims 105, 106, or 108 to 135, wherein the PRP is to be administered to the subject once every month, or once every six months.

137. Use according to any of claims 105, 106, or 108 to 136, wherein the PRP is for intravenous administration to the subject.

138. Use according to any of claims 105, 106, or 108 to 137 wherein the PRP comprises platelets obtained from the subject. 56

139. Use according to any of claims 105, 106, or 108 to 138, wherein the PRP is autologous PRP.

140. Use according to any of claims 105, 106, or 108 to 139, wherein the PRP comprises additional growth factors.

141. Use according to any of claims 105, or 107 to 140, wherein the vitamin A is to be administered to the subject at a maintenance dose of vitamin A, wherein the maintenance dose is less than a full treatment dose.

142. Use according to claim 141 , wherein the maintenance dose is up to three quarters of a full treatment dose.

143. Use according to claim 141 or 142, wherein the maintenance dose is up to two-thirds of a full treatment dose.

144. Use according to any of claims 141 to 143, wherein the maintenance dose is at least a quarter of a full treatment dose.

145. Use according to any of claims 141 to 144, wherein the maintenance dose is to be administered after the subject has been administered one or more full-treatment doses.

146. Use according to any of claims 141 to 145, wherein the maintenance dose is to be administered from the day after the last administration of a full-treatment dose to the subject

147. Use according to any of claims 141 to 146, wherein a plurality of maintenance doses is to be administered to the subject.

148. Use according to claim 147, wherein the maintenance doses are to be administered for at least one week from the day of first administration of a maintenance dose to the subject.

149. Use according to claim 147, wherein the maintenance doses are to be administered for at least 4 weeks from the day of first administration of a maintenance dose to the subject.

150. Use according to any of claims 141 to 149, wherein the PRP is to be administered to the subject at least one week after the last administration of a maintenance dose of vitamin A.

151. Use according to any of claims 141 to 150, wherein the subject is a human subject.

152. Use according to claim 151 , wherein the, or each maintenance dose comprises >2,500 IU to 75,000 IU vitamin A per day. 57

153. Use according to claim 152, wherein the, or each maintenance dose comprises 5,000-75,000, 10,000-75,000, or 20,000-75,000 IU vitamin A per day.

154. Use according to claim 152, wherein the, or each maintenance dose comprises 5,000-50,000, 10,000-50,000, or 20,000-50,000 IU vitamin A per day.

155. Use according to any of claims 141 to 150, wherein the subject is a non-human subject.

156. Use according to claim 155, wherein the subject is a horse.

157. Use according to claim 156, wherein the, or each maintenance dose comprises 10,000-200,000, 20,000-150,000, 40,000-100,000, or 60,000-100,000 IU vitamin A per day.

158. A combined preparation, which comprises: (a) vitamin A; and (b) platelet-rich plasma (PRP).

159. A pharmaceutical composition, which comprises: (a) vitamin A; (b) PRP; and (c) a pharmaceutically acceptable carrier, excipient, or diluent.

160. A combined preparation according to claim 158, wherein the vitamin A is separate from the PRP.

161. A combined preparation according to claim 158 or 160, or a pharmaceutical composition according to claim 159, wherein the vitamin A and the PRP are for coadministration to a subject.

162. A combined preparation according to claim 158 or 160, or a pharmaceutical composition according to claim 159, wherein the vitamin A and the PRP are for sequential administration to a subject.

163. A combined preparation or a pharmaceutical composition according to any of claims

158 to 162, which comprises a plurality of doses of the vitamin A.

164. A combined preparation or a pharmaceutical composition according to any of claims

158 to 163, which comprises a plurality of doses of the PRP.

165. A combined preparation or a pharmaceutical composition according to any of claims

158 to 164, wherein the vitamin A comprises isolated vitamin A.

166. A combined preparation or a pharmaceutical composition according to any of claims 158 to 165, wherein the vitamin A comprises a preformed vitamin A, such as a retinyl ester or retinol. 58

167. A combined preparation or a pharmaceutical composition according to any of claims 158 to 166, wherein the vitamin A comprises a provitamin A, such as a carotenoid.

168. A combined preparation or a pharmaceutical composition according to any of claims 158 to 167, wherein the vitamin A comprises a bioactive form of vitamin A, such as retinal or retinoic acid.

169. A combined preparation or a pharmaceutical composition according to any of claims 158 to 168, wherein the vitamin A is present in a unit dose form which comprises up to 50% of a maximum safe daily dose of vitamin A for a subject per day.

170. A combined preparation or a pharmaceutical composition according to any of claims 158 to 169, wherein the vitamin A is present in a unit dose form which comprises >10% to 50% of a maximum safe daily dose of vitamin A for a subject.

171. A combined preparation or a pharmaceutical composition according to any of claims 158 to 170, wherein the vitamin A is present in a unit dose form which comprises 25% to 50% of a maximum safe daily dose of vitamin A for a subject.

172. A combined preparation or a pharmaceutical composition according to any of claims 158 to 171 , wherein the vitamin A is present in a unit dose form which comprises up to 50% of a minimum toxic dose for a subject.

173. A combined preparation or a pharmaceutical composition according to any of claims 158 to 172, wherein the vitamin A is present in a unit dose form which comprises at least 5% of a minimum toxic dose for a subject.

174. A combined preparation or a pharmaceutical composition according to any of claims 158 to 173, wherein the subject is a human subject.

175. A combined preparation or a pharmaceutical composition according to claim 174, wherein the vitamin A is present in a unit dose form which comprises >10,000 to 100,000 III vitamin A.

176. A combined preparation or a pharmaceutical composition according to claim 175, wherein the vitamin A is present in a unit dose form which comprises about 25,000-100,000, 50,000-100,000, or 75,000-100,000 IU vitamin A.

177. A combined preparation or a pharmaceutical composition according to any of claims 158 to 173, wherein the subject is a horse.

178. A combined preparation or a pharmaceutical composition according to claim 177, wherein the vitamin A is present in a unit dose form which comprises 25,000-250,000, 59

50,000-250,000, 75,000-250,000, 100,000-250,000, 125,000-250,000, 150,000-250,000, 175,000-250,000, or 200,000-250,000 IU vitamin A.

179. A combined preparation or a pharmaceutical composition according to claim 177, wherein the vitamin A is present in a unit dose form which comprises 25,000-50,000, 25, DOO- 75, 000, 25,000-100,000, 25,000-125,000, 25,000-150,000, 25,000-175,000, or 25,000- 200,000 III vitamin A.

180. A combined preparation or a pharmaceutical composition according to any of claims 158 to 179, wherein the vitamin A is for systemic administration to a subject.

181. A combined preparation or a pharmaceutical composition according to claim 180, wherein the vitamin A is for oral or intravenous administration to a subject.

182. A combined preparation or a pharmaceutical composition according to any of claims 158 to 181 , wherein the PRP is present in a unit dose form which comprises a platelet concentration of at least 1 ,000,000 platelets/ l.

183. A combined preparation or a pharmaceutical composition according to any of claims 158 to 182, wherein the PRP is for intravenous administration to a subject.

184. A combined preparation or a pharmaceutical composition according to any of claims 158 to 183, wherein the PRP comprises autologous PRP.

185. A combined preparation or a pharmaceutical composition according to any of claims 158 to 184, wherein the PRP comprises additional growth factors.

186. A combined preparation or a pharmaceutical composition according to any of claims 158 to 185, wherein the vitamin A is present in unit dose form which is a maintenance dose, which is less than a full treatment dose.

187. A combined preparation or a pharmaceutical composition according to claim 186, wherein the maintenance dose is up to three quarters of a full treatment dose.

188. A combined preparation or a pharmaceutical composition according to claim 186 or 187, wherein the maintenance dose is up to two-thirds of a full treatment dose.

189. A combined preparation or a pharmaceutical composition according to any of claims 186 to 188, wherein the maintenance dose is at least a quarter of a full treatment dose.

190. A combined preparation or a pharmaceutical composition according to any of claims 186 to 189, wherein the subject is a human subject. 60

191. A combined preparation or a pharmaceutical composition according to claim 190, wherein the, or each maintenance dose comprises >2,500 III to 75,000 IU vitamin A.

192. A combined preparation or a pharmaceutical composition according to claim 191 , wherein the, or each maintenance dose comprises 5,000-75,000, 10,000-75,000, or 20, DOO- 75, 000 IU vitamin A.

193. A combined preparation or a pharmaceutical composition according to claim 191 , wherein the, or each maintenance dose comprises 5,000-50,000, 10,000-50,000, or 20,000- 50,000 IU vitamin A.

194. A combined preparation or a pharmaceutical composition according to any of claims 186 to 189, wherein the subject is a non-human subject.

195. A combined preparation or a pharmaceutical composition according to claim 194, wherein the subject is a horse.

196. A combined preparation or a pharmaceutical composition according to claim 195, wherein the, or each maintenance dose comprises 10,000-200,000, 20,000-150,000, 40,000-100,000, or 60,000-100,000 IU vitamin A.

197. A combined preparation or a pharmaceutical composition according to any of claims 158 to 196 for use as a medicament.

198. A combined preparation or a pharmaceutical composition according to any of claims 158 to 196 for use in the treatment of a connective tissue injury.

199. Use of a combined preparation or a pharmaceutical composition according to any of claims 158 to 196 in the manufacture of a medicament for the treatment of a connective tissue injury.

200. A combined preparation or a pharmaceutical composition according to claim 198, or use according to claim 199, wherein the connective tissue injury is a tendon injury, or a ligament injury.